Epitope-captured antibody display

ABSTRACT

Reagents and methods for detecting target proteins in a sample are provided. The reagents include a replicable genetic package, a protein displayed on an exterior surface of the package that is expressed from a heterologous nucleic acid borne by the package, and one or more antibodies complexed with the expressed protein and which have an open binding site for a target protein. Thus, a segment of the nucleic acid encodes for an epitope that is shared by the expressed polypeptide and the target protein. The reagents can be utilized individually or as part of a library or an array to bind target proteins within protein samples to form one or more complexes. By determining the sequence of the segment of the heterologous nucleic acid of a package within a complex, one can identify the target protein since the segment encodes for an epitope that is shared by the expressed and target proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/284,305, filed Apr. 17, 2001, which is incorporatedherein by reference in its entirety for all purposes.

BACKGROUND

[0002] One of the major goals of current functional genomics research isto establish correlations between gene expression levels and particularcellular states of interest (e.g., disease states, certain developmentalstages, states associated with exposure to particular environmentalstimuli and states resulting from administration of particulartherapeutic treatments). The establishment of such correlations has thepotential to provide significant insight into the mechanism of disease,cellular development and differentiation, as well as being of value inthe identification of new therapeutics, drug targets and/or diseasemarkers.

[0003] Historically, functional genomic studies have focused on mRNAlevels in making such correlations. This focus is due in large partbecause of the generic nature of the methodology for detecting differentmRNAs, namely the detection of hybridization between nucleic acid probesand target mRNA molecules. Recent research, however, indicates thatoften mRNA expression does not correlate well with protein expression,and even less well with protein accumulation or content. Such resultsare not particularly surprising since many factors affect protein levelsindependent of transcriptional control, including for example,differences in translational efficiency, turnover rates, whether theprotein is compartmentalized or expressed extracellularly, andpost-translational modifications. Thus, profiling proteins rather thanmRNA is often the preferred approach for conducting functional genomicstudies. This is particularly true since proteins are the cellularagents responsible for the catalytic activity of a cell or tissue;hence, by monitoring protein expression, one is able to more directlymonitor the actual agents responsible for the biological processes thatoccur within the cell or tissue.

[0004] Various techniques have been utilized in analyzing the proteincontent of a cell or tissue. Two-dimensional (2-D) gel electrophoresisis one of the more widely utilized techniques for performing suchanalyses. As the name implies, the method involves separating proteinswithin a cell or tissue into two dimensions on an electrophoreticseparation matrix. The separated proteins are then typically detected byvarious staining protocols thus yielding a multitude of spots on thegel. If the separation is done under appropriate conditions, thelocation of the proteins can be used to identify particular proteins, orat least to provide a “fingerprint” of the proteins present inparticular cells. There has been a proliferation of protein gel imagedatabases to assist in the identification and comparison of proteinlevels in different cells and tissues. An example of such a database isthe Protein-Disease Database maintained by the National Institutes ofHealth (NIH). A significant limitation of such methods, however, is thedifficulty in identifying the proteins present at each of the spots on agel.

[0005] Phage-display technology is a technology that has been widelyutilized in protein analysis. However, this technology has been utilizedprimarily to produce and screen large libraries of polypeptides toidentify polypeptides capable of specifically binding to, particulartargets (see, e.g., Cwirla et al., Proc. Natl. Acad. Sci. USA87:6378-6382 (1990); Devlin et al., Science 249:404-406 (1990); Scottand Smith, Science 249:386-388 (1990); and Ladner et al., U.S. Pat. No.5,571,698). Phage display methods typically involve the insertion ofrandom oligonucleotides into a phage genome such that they direct abacterial host to express peptide libraries fused to phage coat proteins(e.g., filamentous phage pIII, pVI or pVIII). Libraries of up to 10¹⁰individual members can be routinely prepared in this way. Incorporationof the fusion proteins into the mature phage coat results in the peptideencoded by the heterologous sequence being displayed on the exteriorsurface of the phage, while the heterologous sequence encoding thepeptide resides within the phage particle.

[0006] The utility of this technology lies in the physical associationbetween the displayed peptide and the genetic material encoding it; thisassociation permits the simultaneous mass screening of very largenumbers of phage bearing different peptides. Phage displaying peptideshaving binding specificity for a particular target can be enriched byaffinity screening against the target. The identity of such peptides canbe determined from the heterologous sequence contained in the phagedisplaying the peptide.

[0007] Display technology can be utilized to prepare recombinantantibody display libraries for use in the analysis of protein samples.Often such libraries are produced as phage display libraries. Conductinganalyses with such libraries is complicated by the fact that in suchlibraries it is the displayed antibody, rather than the target proteinspecifically bound by the antibody, that is encoded by the heterologousnucleic acid sequence within the display package (typically abacteriophage).

[0008] Hence, although various methods for conducting certain types ofprotein analysis have been developed, a significant impediment toanalyzing protein expression as a means to gain insight into biologicalprocesses is the lack of a generic detection reagent and methodologythat is comparable to the ability to use nucleic acid probes inhybridization reactions as detection reagents to detect the presence ofcomplementary nucleic acids.

SUMMARY

[0009] A variety of reagents, arrays of polypeptides and methods areprovided for analyzing and detecting proteins and for studyingprotein/protein interactions. In general, the reagents comprise areplicable genetic package that displays a polypeptide encoded by aheterologous segment of a nucleic acid of the package, and a capturedmultivalent antibody having specific affinity for the displayedpolypeptide which is complexed thereto. Because the captured antibody ismultivalent, in addition to binding to the displayed polypeptide, theantibody has one or more additional binding sites that are available tobind to a target polypeptide that shares an epitope with the displayedpolypeptide. A population of such reagents constitutes a library ofantibodies displayed on replicable genetic packages.

[0010] These reagents disclosed herein are distinctly different fromconventional antibody display libraries. The reagents provided hereinhave a heterologous nucleic acid segment that encodes the target proteinthat becomes complexed with a reagent. With conventional polypeptidedisplay libraries, in contrast, a heterologous nucleic acid segmentencodes the displayed protein rather than the target protein that formsa complex with the displayed protein (antibody). Consequently, thereagents provided herein, utilized either individually or ascollections, can be utilized in a wide variety of methods to detect andidentify polypeptides in samples of a variety of different types (e.g.,solutions, gel matrices such as one- and two-dimensional electrophoreticgels; and tissue samples). The reagents can also be immobilized onarrays to facilitate certain types of analyses.

[0011] Certain methods utilizing such reagents generally involveproviding a population of replicable genetic package/antibody reagentssuch as just described, wherein members of the population comprise areplicable genetic package that displays a first polypeptide encoded bya heterologous segment of a nucleic acid of the package, and the firstpolypeptide is complexed with a captured antibody having specificaffinity for the polypeptide; the first polypeptide and the capturedantibody complexed with it varying between at least some of thepackage/antibody reagents. This population of package/antibody reagentsis contacted with a second polypeptide, whereby package/antibodyreagents bearing captured antibodies having specific affinity for thesecond polypeptide bind to the second polypeptide. At least onepackage/antibody reagent that binds to the second polypeptide isidentified. The sequence of the segment of the nucleic acid of the atleast one package/antibody reagent and its corresponding amino acidsequence is determined to obtain an indication of an epitope shared bythe first and second polypeptides.

[0012] With some methods, a population of immunogens is prepared togenerate a population of antibodies that are then reacted with apopulation of replicable genetic packages to form the package/antibodyreagents. In some instances, the population of immunogens is a displaylibrary, wherein members of the display library include a replicablegenetic package that displays one of the polypeptides displayed by thepackage/antibody reagents. When display libraries are utilized as theimmunogen, generally the replicable genetic package of the displaylibrary is chosen to be of a different type than the replicable geneticpackage of the package/antibody reagents.

[0013] The package/antibody reagents can also be utilized in arrays.Certain arrays include a support and a plurality of polypeptidesimmobilized at different locations on the support, wherein there are atleast 10³ locations/cm² on the support, each location having at leastone of the plurality of polypeptides immobilized therein. Thepolypeptides in at least some of the locations differ in amino acidsequence and/or another property (e.g., post-translational modification)from polypeptides in other locations. In other arrays, the polypeptidesdiffer in amino acid sequence and/or another property in each of thelocations. The polypeptides in certain arrays are antibodies of thepackage/antibody reagents. The polypeptides in other arrays are proteinsthat have been captured by the antibody of package/antibody reagentsthat are immobilized on a support. Some arrays have a higher density oflocations, such as 10⁴, 10⁶, 10⁸ or 10¹⁰ locations/cm², for example. Thearrays can have tens, hundreds, thousands, tens of thousands or hundredsof thousands of different polypeptides immobilized to the support.

[0014] Other arrays include a support and a plurality of polypeptidesimmobilized to the support, at least some of the plurality ofpolypeptides complexed with a captured antibody of a package/antibodyreagent. Each of the package/antibody reagents comprise a replicablegenetic package that displays a polypeptide, which in turn is complexedto the captured antibody. In certain arrays of this type, the support isa gel or a replica of the gel, and the plurality of polypeptides arelocated within the gel or on the replica.

[0015] Arrays of the package/antibody reagents can be used to conduct anumber of different types of analysis. Some methods involve providing anarray comprising a support and a plurality of replicable geneticpackage/antibody reagents immobilized to the support, wherein thepackage/antibody reagents comprise a replicable genetic package thatdisplays a polypeptide encoded by a segment of a nucleic acid of thepackage, and the polypeptide is complexed with a captured antibodyhaving specific affinity for the polypeptide, the polypeptide and themultivalent captured antibody complexed with it varying between at leastsome of the package/antibody reagents. The array is then contacted witha sample containing a mixture of polypeptides, whereby package/antibodyreagents bearing captured antibodies having specific affinity for apolypeptide in the mixture capture the polypeptide from the mixture toform a complex. At least one of the complexes is detected. The sequenceof the segment of the nucleic acid of the package/antibody reagentwithin the at least one complex and the corresponding amino acidsequence provides an indication of an amino acid sequence of an epitopeon the captured polypeptide.

[0016] Methods of preparing various types of arrays are also provided.Certain of these methods involve immobilizing replicable geneticpackages displaying a polypeptide to a support. The displayedpolypeptides are subsequently contacted with a population of antibodiesunder conditions such that the antibodies form complexes with displayedpolypeptides for which the antibodies have specific binding affinity,whereby the array of polypeptides is formed. When the replicable geneticpackages are phage, in some instances the phage are immobilized byplating the phage on a layer of cells to form bacterial microcolonies oran array of micro-plaques. The microcolonies or micro-plaques are thenreplicated onto the support, whereby phage displaying polypeptidesbecome immobilized to the support. Other methods involve an additionalstep in which the array is contacted with a sample containing aplurality of proteins under conditions such that proteins in the sampleand antibodies on the array that have specific binding affinity for oneanother form complexes, thereby forming an array of captured proteins.In some instances, the plurality of proteins in the sample arefunctional proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017]FIG. 1 is a schematic representation of the primary components ofcertain replicable genetic package/antibody reagents.

[0018]FIG. 2 shows the results of an ELISA of T7 phage displaying hTNFRcDNA fragments captured by immobilized goat anti-hTNFR polyclonalantibody.

[0019]FIG. 3 depicts the deduced protein sequences displayed by T7 hTNFRcDNA fragment phage clones selected for binding to the goat anti-hTNFRpolyclonal antibody. The sequences are aligned with the protein sequenceof the hTNFR-1 extracellular domain.

[0020]FIG. 4 shows the results of an ELISA of a T7 hTNFR cDNA fragmentphage clone captured with the polyclonal anti-TNFR antibody toimmobilized hTNFR.

[0021]FIGS. 5A and 5B show the detection of rhTNFR on a Western blotprobed with an anti-hTNFR T7 phage-displayed antibody.

[0022]FIGS. 6A and B illustrate the recovery and enrichment of infectivetarget phage particles from anti-hTNFR T7 phage-displayed antibodycomplexes bound to rhTNFR on a Western blot.

[0023]FIGS. 7A and 7B show the detection of rhTNFR in a complex mixtureof proteins on a Western blot probed with an anti-hTNFR T7phage-displayed antibody.

[0024]FIG. 8 shows the results of an ELISA of fd phage displaying hTNFRcDNA fragments captured by immobilized goat anti-hTNFR polyclonalantibody.

[0025]FIG. 9 depicts the deduced protein sequences displayed by fd hTNFRcDNA fragment phage clones selected for binding to the goat anti-hTNFRpolyclonal antibody. The sequences are aligned with the protein sequenceof the hTNFR-1 extracellular domain.

[0026]FIGS. 10A and 10B show the results of an ELISA of a T7 hTNFR cDNAfragment phage clone captured with the polyclonal anti-TNFR antibody toimmobilized hTNFR.

[0027]FIGS. 11A and B show the detection of rhTNFR on a Western blotprobed with an anti-hTNFR fd phage-displayed antibody.

[0028]FIGS. 12A and 12B show Western blots of lysates from MDCK, NRK49F, NRK 52E, and Caco-2 cells separated by SDS-PAGE and probed withvarious bleeds from 1 of three rabbits inoculated with “live MDCK cellprep” (FIG. 12A) or bleeds from 1 of three rabbits inoculated with“fixed MDCK cell prep” (FIG. 12B) and visualized using ¹²⁵I-labeled goatanti-rabbit secondary Ab.

[0029]FIGS. 13A and 13B show samples of 100K membrane preparations fromdifferentiated MDCK cells (FIG. 13A) and Caco-2 cells (FIG. 13B)separated by SDS-PAGE and visualized by staining the proteins withSyproRuby.

[0030]FIGS. 14A and 14B show Western blots of lysates from MDCK, NRK49F, NRK 52E, and Caco-2 cells separated by SDS-PAGE and probed withvarious bleeds from 1 of three rabbits inoculated with “MDCK 100k memprep” (FIG. 14A) or bleeds from 1 of three rabbits inoculated with“Caco-2 100k mem prep” (FIG. 14B) and visualized using ¹²⁵I-labeled goatanti-rabbit secondary Ab.

[0031] FIGS. 15A-15C illustrate the structures of exemplary arrays asprovided herein. FIG. 15A illustrates an array in which a polypeptide isimmobilized to a support and is complexed with a captured antibody of adetection reagent that includes a replicable genetic package displayinga display polypeptide that is complexed with the captured antibody. Anepitope shared by the immobilized polypeptide and the displayedpolypeptide can be determined from a segment of a heterologous sequencein the replicable genetic package that encodes for the displayedpolypeptide. FIG. 15B shows an array in which package/antibody reagents(each comprising a replicable genetic package displaying a polypeptidethat is complexed with a captured antibody) are immobilized to asupport. An epitope of a polypeptide that binds to the captured antibodycan be determined from a segment of the heterologous nucleic acid of thereplicable genetic package that encodes for the displayed polypeptide.FIG. 15C depicts an array similar to that in FIG. 15B, but the compleximmobilized to the support also includes a polypeptide captured by theantibody of the package/antibody reagent. The captured polypeptides inthese arrays can be assayed for activity and/or utilized in studies ofprotein/protein interactions. The identity of an epitope of the capturedpolypeptide can be determined as described with respect to FIG. 15B.

DESCRIPTION

[0032] I. Definitions

[0033] The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide”are used interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer in either single-, double, or triple-strandedform. For the purposes of the present disclosure, these terms are not tobe construed as limiting with respect to the length of a polymer. Theterms can encompass known analogues of natural nucleotides, as well asnucleotides that are modified in the base, sugar and/or phosphatemoieties. In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T. The terms additionally encompass nucleic acids containing knownnucleotide analogs or modified backbone residues or linkages, that aresynthetic, naturally occurring, and non-naturally occurring and thathave similar binding properties as the reference nucleic acid. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0034] A “probe” is an nucleic acid capable of binding to a targetnucleic acid of complementary sequence through one or more types ofchemical bonds, usually through complementary base pairing, usuallythrough hydrogen bond formation, thus forming a duplex structure. Theprobe binds or hybridizes to a “probe binding site.” The probe can belabeled with a detectable label to permit facile detection of the probe,particularly once the probe has hybridized to its complementary target.The label attached to the probe can include any of a variety ofdifferent labels known in the art that can be detected by chemical orphysical means, for example. Suitable labels that can be attached toprobes include, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, and enzyme substrates. Probes canvary significantly in size. Some probes are relatively short. Generally,probes are at least 7 to 15 nucleotides in length. Other probes are atleast 20, 30 or 40 nucleotides long. Still other probes are somewhatlonger, being at least 50, 60, 70, 80, 90 nucleotides long. Yet otherprobes are longer still, and are at least 100, 150, 200 or morenucleotides long. Probes can be of any specific length that falls withinthe foregoing ranges as well.

[0035] “Polypeptide” and “protein” are used interchangeably herein andinclude a molecular chain of amino acids linked through peptide bonds.The terms do not refer to a specific length of the product. Thus,“peptides,” “oligopeptides,” and “proteins” are included within thedefinition of polypeptide. The terms include post-translationalmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. In addition, proteinfragments, analogs, mutated or variant proteins, fusion proteins and thelike are included within the meaning of polypeptide. A “target protein”refers to a protein in a sample whose presence is to be detected.

[0036] “Conservatively modified variations” or simply “conservativevariations” and other similar terms when used to refer to a particularamino acid sequence refer to substitution of amino acids with otheramino acids having similar properties (e.g., acidic, basic, positivelyor negatively charged, polar or non-polar, etc.) such that thesubstitutions of even critical amino acids do not substantially alteractivity. Conservative substitution tables providing functionallysimilar amino acids are well-known in the art. See, e.g., Creighton(1984) Proteins, W. H. Freeman and Company. The terms also refer toindividual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids in anencoded sequence.

[0037] An “exogenous molecule” is a molecule that is not normallypresent in a cell, but can be introduced into a cell by one or moregenetic, biochemical or other methods. Normal presence in the cell isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

[0038] An exogenous molecule can be, among other things, a smallmolecule, such as is generated by a combinatorial chemistry process, ora macromolecule such as a protein, nucleic acid, carbohydrate, lipid,glycoprotein, lipoprotein, polysaccharide, any modified derivative ofthe above molecules, or any complex comprising one or more of the abovemolecules. Nucleic acids include DNA and RNA, can be single- ordouble-stranded; can be linear, branched or circular; and can be of anylength. Nucleic acids include those capable of forming duplexes, as wellas triplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251.

[0039] An exogenous molecule can be the same type of molecule as anendogenous molecule, e.g., protein or nucleic acid (i.e., an exogenousgene), providing it has a sequence that is different from an endogenousmolecule. For example, an exogenous nucleic acid can comprise aninfecting viral genome, a plasmid or episome introduced into a cell, ora chromosome that is not normally present in the cell. Methods for theintroduction of exogenous molecules into cells are known to those ofskill in the art and include, but are not limited to, lipid-mediatedtransfer (i.e., liposomes, including neutral and cationic lipids),electroporation, direct injection, cell fusion, particle bombardment,calcium phosphate co-precipitation, DEAE-dextran-mediated transfer andviral vector-mediated transfer.

[0040] By contrast, an “endogenous molecule” is one that is normallypresent in a particular cell at a particular developmental stage underparticular environmental conditions.

[0041] A “heterologous sequence” or a “heterologous nucleic acid,” isone that originates from a source foreign to the particular replicablegenetic package, or, if from the same source, is modified from itsoriginal form. Thus, a heterologous gene in a prokaryotic replicablegenetic package includes a gene that, although being endogenous to theparticular host replicable genetic package, has been modified.Modification of the heterologous sequence can occur, e.g., by treatingthe DNA with a restriction enzyme to generate a DNA fragment that iscapable of being operably linked to the promoter. Techniques such assite-directed mutagenesis are also useful for modifying a heterologousnucleic acid.

[0042] The term “operably linked” refers to functional linkage between anucleic acid expression control sequence (such as a promoter, signalsequence, or array of transcription factor binding sites) and a secondpolynucleotide, wherein the expression control sequence affectstranscription and/or translation of the second polynucleotide.

[0043] The term “recombinant” when used with reference to a cellindicates that the cell replicates a heterologous (exogenous) nucleicacid, or expresses a peptide or protein encoded by a heterologous(exogenous) nucleic acid. Recombinant cells can contain genes that arenot found within the native (non-recombinant) form of the cell.Recombinant cells can also contain genes found in the native form of thecell wherein the genes are modified and re-introduced into the cell byartificial means. The term also encompasses cells that contain a nucleicacid endogenous to the cell that has been modified without removing thenucleic acid from the cell; such modifications include those obtained bygene replacement, site-specific mutation, and related techniques.

[0044] A “fusion molecule” is a molecule in which two or more subunitmolecules are linked, generally covalently. The subunit molecules can bethe same chemical type of molecule, or can be different chemical typesof molecules. Examples of the first type of fusion molecule include, butare not limited to, fusion polypeptides and fusion nucleic acids.

[0045] The term “antibody” as used herein includes antibodies of anymultivalent form, including multiple copy display of monovalentantibodies, such as found in replicable genetic packages (e.g., phage)that display scFv fragments. A “multivalent” antibody refers to anantibody that is capable of binding multiple copies of a single antigenor that is capable of binding to two or more different antigens thatshare an epitope. The antibodies can be obtained from both polyclonaland monoclonal preparations. An antibody consists of one or morepolypeptides substantially encoded by immunoglobulin genes or fragmentsof immunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

[0046] A typical immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chains,respectively.

[0047] Antibodies exist as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)2 dimer into anFab′ monomer. The Fab′ monomer is essentially an Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments can be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies, more preferably single chain Fv (scFv) antibodies in which avariable heavy and a variable light chain are joined together (directlyor through a peptide linker) to form a continuous polypeptide.

[0048] A single chain Fv (“scFv”) polypeptide is a covalently linkedVH::VL heterodimer which can be expressed from a nucleic acid includingVH- and VL-encoding sequences either joined directly or joined by apeptide-encoding linker. Huston, et al. Proc. Nat. Acad. Sci. USA,85:5879-5883 (1988). A number of strategies for converting the naturallyaggregated—but chemically separated light and heavy polypeptide chainsfrom an antibody V region into an scFv molecule that will fold into athree dimensional structure substantially similar to the structure of anantigen-binding site, have been reported. See, e.g. U.S. Pat. Nos.5,091,513 and 5,132,405 and 4,956,778. Antibodies can also be diabodies,tribodies and tetrabodies.

[0049] An “antigen-binding site” or “binding portion” refers to the partof an immunoglobulin molecule that participates in antigen binding. Theantigen-binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains are referred to as “hypervariable regions” which are interposedbetween more conserved flanking stretches known as “framework regions”or “FRs”. Thus, the term “FR” refers to amino acid sequences that arenaturally found between and adjacent to hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen binding “surface”. This surface mediates recognition andbinding of the target antigen. The three hypervariable regions of eachof the heavy and light chains are referred to as “complementaritydetermining regions” or “CDRs” and are characterized, for example byKabat et al. Sequences of proteins of immunological interest, 4th ed.U.S. Dept. Health and Human Services, Public Health Services, Bethesda,Md. (1987).

[0050] The term “epitope” refers to the portion of an antigen thatinteracts with an antibody. More specifically, the term epitope includesany protein determinant capable of specific binding to an immunoglobulinor T-cell receptor. The phrase “shared epitope” and other relatedphrases means that two or more polypeptides present an epitope that isspecifically recognized by the same antibody. In some instances theamino acid sequence of the epitope of such polypeptides is identical; inother instances, however, the amino acid sequence of the epitopepresented by the polypeptides varies slightly, in some instances by onlyone or two amino acids. The phrase can also refer to continuous ordiscontinuous epitopes in which the primary sequence (i.e., the aminoacid sequence) is not similar but nonetheless the epitopes are stillrecognized by the same antibody.

[0051] The phrases “specifically binds,” “specific binding affinity” (orsimply “specific affinity”), “specifically recognize,” and other relatedterms when used to refer to binding between a protein and an antibody,refers to a binding reaction that is determinative of the presence ofthe protein in the presence of a heterogeneous population of proteinsand other biologics. Thus, under designated conditions, a specifiedantibody binds preferentially to a particular protein and does not bindin a significant amount to other proteins present in the sample. Anantibody that specifically binds to a protein has an associationconstant of at least 10³ M⁻¹ or 10⁴ M⁻¹, sometimes 10⁵ M⁻¹ or 10⁶ M⁻¹,in other instances 10⁶ M⁻¹ or 10⁷ M⁻¹, preferably 10⁸ M⁻¹ to 10⁹ M⁻¹,and more preferably, about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher. A variety ofimmunoassay formats can be used to select antibodies specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See, e.g., Harlow and Lane(1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,New York, for a description of immunoassay formats and conditions thatcan be used to determine specific immunoreactivity.

[0052] Reference to a polypeptide being “displayed” on a replicablegenetic package means that the polypeptide is attached to a group (e.g.,an amino acid residue) located at an exterior surface of the replicablegenetic package.

[0053] An “array” broadly refers to an arrangement of agents (e.g.,proteins, antibodies, replicable genetic packages) in positionallydistinct locations on a substrate. In some instances the agents on thearray are spatially encoded such that the identity of an agent can bedetermined from its location on the array. A “microarray” generallyrefers to an array in which detection requires the use of microscopicdetection to detect complexes formed with agents on the substrate. A“location” on an array refers to a localized area on the array surfacethat includes agents, each defined so that it can be distinguished fromadjacent locations (e.g., being positioned on the overall array, orhaving some detectable characteristic, that allows the location to bedistinguished from other locations). Typically, each location includes asingle type of agent but this is not required. The location can have anyconvenient shape (e.g., circular, rectangular, elliptical orwedge-shaped). The size or area of a location can vary significantly. Insome instances, the area of a location is greater than 1 cm², such as2-20 cm², including any area within this range. More typically, the areaof the location is less than 1 cm², in other instances less than 1 mm²,in still other instances less than 0.5²mm, in yet still other instancesless than 10,000 μm², or less than 100 μm.

[0054] An “electrophoretic separation matrix” refers to any matrix inwhich components of a sample are electrophoretically separated.Typically, components include a plurality of polypeptides in apolypeptide sample which is being analyzed. Such matrices can includevarious types of solutions and gels. Examples of such matrices include,but are not limited to, polyacrylamide, agarose and cellulose.

[0055] A “tissue” refers to an aggregation of cells united inperformance of a particular function. The tissue can be part of a livingorganism, a section excised from a living organism, or can beartificial. An artificial tissue is one in which the aggregation ofcells are grown to function similar to a tissue in a living organism.The aggregated cells, however, are not obtained from a host (i.e., aliving organism). Artificial tissues can be grown in vivo or in vitro.

[0056] A “label” refers to an agent that can be detected by usingphysical, chemical, optical, electromagnetic and/or other methods.Examples of detectable labels that can be utilized include, but are notlimited to, radioisotopes, fluorophores, chromophores, mass labels,electron dense particles, magnetic particles, spin labels, moleculesthat emit chemiluminescence, electrochemically active molecules,enzymes, cofactors, and enzyme substrates.

[0057] II. Overview

[0058] Described herein are methods and reagents that can be utilized toanalyze the composition of complex protein mixtures, including detectingthe presence of one or more particular proteins in a mixture ofproteins. More specifically, the reagents include a replicable geneticpackage (or simply package) having a heterologous nucleic acid thatencodes a polypeptide that is displayed on an exterior surface of thereplicable genetic package. The displayed polypeptide is complexed to anantibody (or plurality of antibodies) that has specific binding affinityfor the displayed polypeptide. Thus, the general overall structure ofthe reagent is package (carrying the heterologous nucleic acidsegment)/expressed protein/captured antibody (see FIG. 1). The antibodyor antibodies complexed to the replicable genetic package are able tobind a plurality of polypeptides. Thus, while some of the binding sitesof the multivalent antibody are bound to the expressed polypeptide, theother site(s) on the antibody (antibodies) is (are) available to bind toa polypeptide (the target polypeptide) that shares an epitope with theexpressed polypeptide. In many instances, the expressed polypeptide andthe polypeptide bound by the complexed antibody are the same (i.e., havethe same primary sequence). In other instances, the epitope on theexpressed polypeptide and the polypeptide bound by the antibody haveslightly different amino acid sequences. Such differences often involvevariation in only one or two amino acids. In some instances, thedifferences involve conservative variations.

[0059] Hence, a population of such reagents constitutes a library ofantibodies displayed on replicable genetic packages. The significance ofsuch a library is that each replicable genetic package of the librarycarries a heterologous nucleic acid segment that encodes an amino acidsequence that correlates with the epitope recognized by the antibodydisplayed on the package. Said differently, each package in such alibrary is a ligand of the protein encoded by the heterologous nucleicacid carried by the package. As just described, this is the case becausethe open binding site(s) on the displayed antibody (antibodies) can bindto one or more additional copies of a protein having a epitope that isshared with the protein expressed on the replicable genetic package, andoften binds to a protein having the same primary sequence. A library ofreagents having this arrangement can be considered an “anti-proteome,”because the reagents within the library can specifically bind to all ora portion of the proteins within a cell or tissue of interest. The powerof such an arrangement is that it permits the immediate identification(by primary sequence) of target proteins in a wide variety of sampletypes, including, but not limited to, gels, cells, tissues and proteinarrays.

[0060] Such reagents are distinctly different from the complexes inconventional recombinant antibody display libraries. With the presentreagents, the heterologous nucleic acid segment carried by the packageencodes the target protein; in contrast, with conventional displaylibraries, the segment encodes the displayed antibody rather than theprotein complexed to the antibody. Consequently, the individual reagentsand libraries disclosed herein can be utilized as a general reagent in awide variety of methods for determining the identity of polypeptides,even in complex mixtures. As such, the reagents have broad applicabilityto protein biochemistry and cell biology, as well as functional genomicsand proteomics.

[0061] For example, the reagents can be utilized to determine theprimary sequence of any, and in some instances all, of the proteins insamples obtained from tissues, cells or subcellular compartments. Thereagents can also be utilized to identify proteins in one- andtwo-dimensional separation matrices, thereby significantly expanding theutility of such established analytical techniques. The reagents can alsobe used in preparing and subsequently utilizing a wide variety ofprotein arrays, including arrays having unknown proteins and arrayshaving known proteins in unknown locations.

[0062] Additionally, the ability to utilize the reagents to obtainqualitative and quantitative information means that the methods areamenable to a variety of screening, comparative and diagnostic studies.For example, the methods can be utilized to develop comparative proteinexpression data. Such comparative studies can be utilized to identifymarkers of specific diseases, potential targets for pharmaceuticalsand/or drug candidates. Once markers that are selectively expressed incertain disease states, for example, are identified, the methods andreagents can be utilized to conduct diagnostic applications.Additionally, the methods and reagents can be used to develop proteindatabases that include, for example, identity and relative abundanceinformation for proteins in different cells, tissues or cellular states.Thus, the methods and reagents can be utilized in differentialexpression analyses. The methods and reagents also find utility instudies on structure/activity relationships and in metabolic engineeringinvestigations in which one genetically modifies a certain gene and thendetermines what effects such a modification has on cellular proteinexpression. The reagents can additionally be used to preparemicroarrays, that in turn can be utilized in both qualitative andquantitative analyses.

[0063] III. Reagent Preparation and Use

[0064] A. Reagent Composition

[0065] As illustrated in FIG. 1, the replicable genetic package/antibodyreagents (or simply “package/antibody reagents or reagents) in generalinclude: (i) a replicable genetic package, (ii) a polypeptidedisplayed/expressed by the replicable genetic package, (iii) aheterologous nucleic acid that includes a segment that encodes thedisplayed polypeptide, and (iv) one or more antibodies (“capturedantibodies”) having specific affinity for, and complexed to, thedisplayed polypeptide. The replicable genetic packages often areutilized as libraries in which members of the library differ withrespect to the displayed polypeptide and the antibody (or antibodies)that are complexed to the displayed polypeptide.

[0066] 1. Replicable Genetic Packages

[0067] Replicable genetic packages (or simply packages) of various typescan be utilized in the package/antibody reagents. In general, areplicable genetic package refers to a biological complex comprising anucleic acid, and at least one peptide encoded by the nucleic acid.Examples of replicable genetic packages include cells, spores, bacteria,viruses, bacteriophage and polysomes. Replicable genetic packages arealso capable of replication either by self-replication, in combinationwith a host and/or a helper virus, or by in vitro replication,transcription and expression. The replicable genetic package can beeither prokaryotic or eukaryotic. Collections of package/antibodyreagents can be selected from any one of the foregoing and includedifferent combinations thereof.

[0068] Bacteriophage including phagemids are often utilized as thereplicable genetic package, especially filamentous phage (e.g., M13, fdand fl) and phagemid vectors derived therefrom. See, e.g., Dower, WO91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047; Huse, WO92/06204; and Kang, WO 92/18619. Other phage of E. coli, such as T7phage, or phage of other bacterial species can also be used. Filamentousphage are generally 6 nm in diameter and up to one micron in length.Such phage have been used extensively in peptide phage display. Thesurface of such phage consists of five coat proteins, two of which, pIIIand pVIII, have been used to display peptide libraries. pIII contains406 amino acids and is present in three to five copies. The major coatprotein, pVIII, which contains 50 amino acids, constitutes the bulk ofthe phage protein as it is present in approximately 2700 copies. Thebacteriophage can also be a non-filamentous phage such as icosahedralphages T7 and lambda. The major coat protein of T7 phage is the gene 10capsid protein, which contains 370 amino acids and is present in 415copies.

[0069] In addition to phage, the replicable genetic package of theinvention can include eukaryotic viruses, (e.g., the Moloney murineleukemia virus; see, e.g., Han, et al. (1995) Proc. Natl. Acad. Sci. USA92:9747-9751) or spores (e.g., spores from B. subtilis; see, e.g.,Donovan, et al. (1987) J. Mol. Biol. 196:1-10). A variety of differentcells can also be used as replicable genetic packages in the presentinvention. Examples of suitable bacterial cells include, but are notlimited to, Salmonella typhimurium, Bacillus subtilis, Pseudomonasaeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseriagonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxellabovis, and especially Escherichia coli.

[0070] Other replicable genetic packages are polysomes. A polysome is acombination of a segment of mRNA with ribosomes attached to the mRNA,the ribosomes also binding a segment of a nascent polypeptide extendingfrom the ribosomes. Such complexes are similar to phage displayparticles in that a heterologous nucleic acid segment that encodes apolypeptide and the polypeptide encoded by the segment are attached toone another. Polysomes are discussed further, for example, in U.S. Pat.No. 5,922,545.

[0071] In some instances, replicable genetic packages are considered tobe of different types when the packages are of different classes, suchas a virus versus a spore, or a virus versus a cell. Replicable geneticpackages can also be of different types even if from the same class butof a different subclass. For example, different filamentousbacteriophage can be considered to be different types (e.g., M13 versusFd versus fl). Different Fd phage displaying different polypeptides,however, would not be considered different types of packages because inboth instances the package is an Fd phage. Different types of replicablegenetic packages can be selected such that the different types are notimmunologically cross reactive (i.e., antibodies that specifically bindto one type of package do not also complex with packages of anothertype).

[0072] 2. Displayed Polypeptide

[0073] The polypeptide expressed and displayed by the replicable geneticpackage can vary widely and can include any length capable of beingdisplayed on a replicable genetic package, including both fragments andfull length proteins, for example. The size of the displayed polypeptidedepends in part on the number of different epitopes one wants displayedby the package. In general, the displayed polypeptide can include justenough amino acids to present a single epitope but can extend in size upto a full length expressed cDNA. Thus, for example, the displayedpolypeptides can include as few as 4, 5 or 6 amino acid residues butextend up to hundreds or even thousands of amino acid residues. Thedisplayed polypeptides in certain applications include at least 4, 5 or6 amino acid residues, and less than 100 residues, but other sizedpolypeptides can be displayed. In some instances, the displayedpolypeptides are 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 residues inlength, or any integral number of amino acids within these ranges.Typically, the displayed polypeptide includes 6 to 20 amino acids. Byutilizing libraries in which the expressed polypeptide is 6-10 aminoacids in length, one can generate libraries having continuous epitopes(i.e., epitopes lacking significant secondary or conformationalstructure resulting from interactions between amino acids in thepolypeptide). Libraries displaying larger polypeptides can be utilizedto generate conformational or discontinuous epitopes that do havesecondary structure.

[0074] Certain package/antibody reagents display random populations ofpeptides. Such libraries are typically designed to produce packages thatdisplay polypeptides in which some or all of the positions of thepolypeptide are systematically varied for the different amino acids.Random peptide coding sequences can be formed by cloning and expressionof randomly-generated mixtures of nucleic acids in the appropriaterecombinant vectors (see, e.g., Oliphant et al. (1986) Gene 44:177-183).Other libraries are formed by producing variants from a startingframework polypeptide. In this approach, a starting polypeptide isutilized as a framework and selected residues are varied. Suchpolypeptides can be formed by mutagenizing the starting nucleic acid byinsertion of mutagenic cassettes or error-prone PCR, for example (see,Lardner et al., WO 88/06630).

[0075] 3. Antibody Complexed to Displayed Polypeptide (“CapturedAntibody”)

[0076] The antibody or antibodies complexed to the displayed polypeptidecan be any multivalent form. As used herein, the term “multivalent”antibody means that the antibody or antibodies can form a complex withthe polypeptide displayed on the replicable genetic package and one ormore copies of a target protein. Thus, the antibody can be a singleantibody with a plurality of binding sites. Alternatively, the capturedantibody or antibodies can include monovalent antibodies, provided suchantibodies are displayed in a multivalent format. One such example is aplurality of monovalent antibodies that are captured by a multivalentantibody. Thus, for example, multiple scFv antibodies can be captured onthe prongs of another antibody (e.g., a scFv captured on an IgGantibody). Another example of multivalent display of a monovalentantibody is the display of scFvs on phage, as phage present multivalentdisplay. The antibodies can also be diabodies, tribodies andtetrabodies.

[0077] The antibodies can be polyclonal populations of immunoglobulinsfrom any convenient species, monoclonal antibodies (derived from micefor example) and recombinant antibody populations as produced in displaysystems such as phage display and diabodies.

[0078] B. Reagent Preparation

[0079] The replicable genetic package/antibody complexes can be formedaccording to a number of different protocols depending upon the startingcomponents utilized. The following steps, however, illustrate oneapproach for preparing the package/antibody reagents beginning withreplicable genetic packages that do not yet contain a heterologoussequence. In general, the process involves: (i) producing a cDNA displaylibrary by preparing a population of replicable genetic packages thateach display different polypeptides; (ii) preparing an antibodypopulation; and (iii) and incubating the antibody population with thecDNA display library under conditions such that antibodies that havespecific binding affinity for a displayed protein form a complex.

[0080] 1. Preparation of cDNA Display Library

[0081] One of the initial steps involves preparing cDNA molecules from acell or tissue of interest and subsequently cloning these cDNA moleculesor fragments thereof into the genome of a replicable genetic package toproduce a library of displayed polypeptides that is at least partially,if not completely, representative of the expressed polypeptidepopulation of the starting cells or tissue. In particular, expression ofthe resulting nucleic acid fusion generates a fusion protein composed ofthe polypeptide encoded by the heterologous segment and an endogenousprotein which, upon its transport and assembly at an outer surface ofthe package, results in the display of an exogenous polypeptide from anexterior surface of the package.

[0082] Thus, for example, nucleic acid libraries frequently are clonedinto the genes for pIII or pVIII using standard cloning techniques toform a fusion gene. Expression of the resulting construct produces afusion protein that includes the display protein, the pIII or pVIIIprotein or fragment thereof, and a signal sequence (typically from asecreted protein). Often the heterologous sequences are inserted into ornear the N-terminus of the gene encoding pIII or pVIII, but this is notrequired and other sites can be utilized. The heterologous nucleicacids, including optional flanking spacers, are inserted into the genomeutilizing established recombinant techniques (see, e.g., Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory Press, Cold Spring Laboratory, N.Y.) and according tothe methods disclosed in the other references listed supra in thissection.

[0083] Certain filamentous phage vectors can be designed to producemultiple copies of either the gene for pIII or pVIII. With such vectors,heterologous nucleic acids are inserted into only one of the copies;expression of the other copy dilutes the proportion of fusion proteinincorporated into the phage and can be useful in reducing selectionagainst polypeptides that reduce phage growth. Another techniqueinvolves cloning the heterologous sequences into phagemid vectors thatencode phage coat proteins and include packaging sequences but which areincapable of self-replication. Such phagemids can be transfected intocells that are also infected with helper phage that package thephagemids (see, e.g., Garrard, WO 92/09690). In certain other instances,a T7 vector is utilized to display 10-20 copies of the polypeptideexpressed by the inserted nucleic acid (i.e., cDNA), which correspondsto approximately one copy of the displayed polypeptide on each face ofthe icosahedral phage particle.

[0084] The cDNA molecules or expressed sequence tags (ESTs) cloned intothe genome of the replicable genetic packages can be prepared by mostconventional means. The present methods do not require full length cDNAbecause function is not required, only that each fragment besufficiently long to contain at least one epitope (see sizeconsiderations listed supra). Often it is desired that most of the cDNAsare large; however, in other instances, a library of smaller cDNAfragments, each containing relatively fewer epitopes per clone, isuseful.

[0085] In general, cDNA preparation methods that do not systematicallyfavor the isolation of any particular region of the cDNA(randomly-primed reverse transcription, for example) are preferred. Thisincreases the diversity of epitopes available in the library. In someinstances, normalizing the cDNA population (see infra) to decrease thedifference in the representation of the most and least abundant mRNAs isdesired, although usually not required. In addition, cDNA segments canbe constructed by oligonucleotide synthesis, from expressed sequencetags listed in publicly or privately available sequence databases. Auniversal human EST library can be constructed to create a universalhuman anti-proteome. Libraries of random peptides can also be used asthe antibody capture reagent (see the following section).

[0086] 2. Antibody Preparation

[0087] Numerous formats are available to prepare the antibodies that arecomplexed to the displayed protein(s) expressed on the exterior surfaceof the replicable genetic packages. In general, one prepares acollection of polypeptides and utilizes the collection as the immunogensto produce the desired antibodies. In some instances, the proteincollection is used to immunize an animal (e.g., a rabbit) to produce apolyclonal antibody collection. Alternatively, the protein collectioncan be used in conjunction with established hybridoma technology togenerate a population of monoclonal antibodies. Still another option isto prepare the antibodies as part of recombinant display libraries.

[0088] Methods for preparing antibodies are discussed, for example, inKohler, G. et al. (1975) Nature 256:495-496; Clausen H. et al. (1985)Biochem. 24:6190-6194; Harlow and Lane (1988) Antibodies: A LaboratoryManual. Cold Spring Harbor Laboratory, New York; and Goding, J. W.(1983) “Monoclonal Antibodies: Principles and Practice, Production andApplication of Monoclonal Antibodies,” in Cell Biology, Biochemistry andImmunology, Academic Press Inc., Victoria, pp. 56-86).

[0089] The population of proteins utilized as the immunogen can be of avariety of different types. One option is to obtain a protein fractionfrom the same cells or tissue utilized to generate the cDNA moleculesused to prepare the cDNA display library. This protein fraction cancontain all of the proteins expressed in the cells or tissues, or somesubset thereof. Using such a collection of proteins, one obtains apopulation of antibodies that are reactive with all or most of theproteins within the cells or tissue of interest.

[0090] Another option is to utilize a display library such as the cDNAdisplay library just described supra as the immunogen in any of theantibody producing formats that are available. Thus, the display libraryused to immunize an animal displays the same or many of the polypeptidesdisplayed by the cDNA library. In some instances, the replicable geneticpackages of the display library utilized as the immunogen are of thesame type as in the cDNA library. More typically, however, thereplicable genetic packages of the display library are of a differenttype then those of the cDNA library. The use of different types ofreplicable genetic packages (e.g., different types of phage) minimizesthe potential problem of generating antibodies against the replicablegenetic packages which could subsequently complex to the replicablegenetic package in the cDNA library rather than the polypeptidedisplayed on the package.

[0091] Thus, for example, a display library in which the polypeptidesare displayed on filamentous phage can be used as the immunogen toproduce an antibody population reactive with the polypeptides displayedon the filamentous phage. This antibody population can then be incubatedwith a cDNA display library in which the same display polypeptides areexpressed on T7 phage under conditions such that the displayed proteinson the T7 phage capture the antibodies generated using the filamentousphage display library as the immunogen.

[0092] The immunizing library can also be expressed as part of a fusionlibrary, such as part of a fusion library to glutathione-S-transferase(GST), for example (see Example 7 infra). For instance, the cDNA used togenerate the cDNA display library can additionally be inserted into avector such that the cDNA is expressed as part of a fusion to GST. Theresulting fusion proteins can be isolated on a glutathione column andthen eluted. The eluted fusion proteins can then be utilized asimmunogens. Alternatively, the fusion proteins can be complexed withbeads that bear glutathione and the resulting beads bearing the fusionproteins utilized as an immunogen, as such bead-borne immunogens havesometimes been found to be more immunogenic.

[0093] Other methods involve cloning random peptide coding sequencesinto appropriate vectors to form a random population of peptides (see,e.g., Oliphant et al. (1986) Gene 44:177-183). The displayedpolypeptides in certain of these random libraries are 6-10 residues inlength but can be up to 20 to 50 or more residues in length. Such alibrary can be considered an “universal immunogen.” Antibodies producedfrom such a display library can then be captured by polypeptides thatare displayed as part of a cDNA library prepared from a cell type ortissue of interest, with the replicable genetic packages of the cDNAlibrary typically differing in type from those used to create the randomdisplay library. As noted supra, however, the replicable geneticpackages of the immunogen and the cDNA display library can in someinstances be of the same type.

[0094] 3. Combining cDNA Display Library and Antibodies

[0095] By mixing the cDNA display library with the population ofantibodies, a library of replicable genetic package/antibody reagents isformed. Conditions for this step are chosen to encourage the formationof complexes between the antibodies and the replicable genetic packages.Generally the density of the displayed polypeptides is low (e.g.,approximately 1 copy per 1000 nm² of package surface). Consequently,even though the antibodies are multivalent (see supra), typically eachantibody only binds to a single copy of the polypeptide displayed on theexterior surface of the package. This leaves the other binding site(s)on the antibody free to bind to a target polypeptide in a sample thatshares an epitope with the polypeptide displayed on the surface. Thus,for example, in the case of libraries in which the packages are T7, eachcaptured antibody typically has at least one free binding site.

[0096] Icosahedral phage vectors are chosen to display approximately 1copy of the expressed polypeptide per phage particle, or 10 to 20 copiesper phage particle (approximately 1 copy of the expressed polypeptideper face of the icosahedron), hence there are approximately 1, or from10 to 20, respectively, free antibody binding sites (per phage) that areavailable for binding to target polypeptides in solution. Mixing of theT7 cDNA library with the antibody population generates a library of T7phage that each display a polypeptide (i.e., display polypeptides) andan antibody or antibodies that have specific binding affinity to variousepitopes on each of the displayed polypeptides.

[0097] The antibodies borne by any particular package can sometimes be apopulation of different antibodies. For example, the antibodiescomplexed to a package can be a collection of different antibodies toany single epitope. Moreover, in some instances, a package may bear aplurality of antibodies recognizing several different epitopes displayedby the expressed polypeptide. The diversity of the antibodies can becontrolled to some extent by the size, and therefore the number ofpotential epitopes encoded, of the displayed polypeptides.

[0098] With other applications, such as applications requiring thehighest of antibody specificities, a single polypeptide rather than aplurality of polypeptides is displayed on the surface of the replicablegenetic package (a monovalent display format; this format in which asingle polypeptide is displayed on a package should not be confused witha monovalent antibody which is an antibody that has a single bindingsite). Examples of monovalent formats that can be utilized include, butare not limited to, fd phagemid pIII vectors, T7 low level expressionvector, and some polysome/ribosome display formats. cDNA productsdisplayed in a monovalent format favor the capture of the highestaffinity (and typically more specific) antibodies. Such reagents placethe highest stringency on binding of the captured antibodies to theimmobilized target proteins; increased stringency also disfavorscross-reactivity of displayed antibodies with polypeptide sequences thatdiffer only slightly from the principle antigenic epitopes.

[0099] Under certain conditions, an antibody or antibodies on onepackage can become specifically cross-linked to polypeptides displayingthe same epitope(s) on other packages, thereby forming epitope-specificaggregates. Such aggregates can be useful in some applications, such asapplications requiring higher antibody valency. Aggregation is usuallyfavored by extending the incubation time to allow like complexes tospecifically cross-react.

[0100] As indicated above, the antibodies of the reagent typically aremultivalent but can be monovalent if displayed in a multivalent format.One example of how a library of such reagents can be prepared is asfollows. Initially, a population of proteins is used to immunize ananimal (e.g., a mouse) to generate a population of antibodies asdescribed above. The spleen of the animal is then removed, mRNA isextracted, reverse transcription and amplification of heavy and lightantibody chain cDNAs is performed. The resulting heavy and light chainfragments are cloned into the appropriate antibody display vectors toexpress single chain Fv or Fab forms on the phage coat proteins, therebycreating a library of many Ab specificities. These two libraries can bemixed to form a library in which monovalent scFv antibodies aredisplayed in a multivalent format. A separate cDNA library of theexpression products of a tissue of interest is also prepared; and thetwo libraries are mixed to form the epitope-captured antibody displaylibrary. Typically, the phage in the antibody library differ in typefrom those of the target tissue cDNA library (e.g., filamentous phageversus T7 phage). In this format, recovery of the bound complex providesclones of the target cDNA and of the target-specific antibodies; thusallowing the identification of the sequences of both the cDNA and theantibodies.

[0101] The size of the library in terms of number of members can varysignificantly. In fact, the reagents can be utilized individually. Forexample, individual reagents can be prepared according to the foregoingmethods that have specific binding affinity for any particularpolypeptide of interest, provided at least some portion of the primarysequence of the polypeptide is known. Many applications, however,utilize libraries or panels of reagents, each member being specific forone of the many individual polypeptides in a tissue, cell or othercomplex mixture of polypeptides, even if some or all of the polypeptidesare of unknown primary sequence. Smaller libraries designed to detectthe presence of a limited number of target polypeptides often have 5-10members, and sometimes more, such as 20, 30, 40 or 50 members, or anyintegral number therebetween. Larger libraries that are utilized toprobe complex protein samples can include a very large number ofmembers, such as 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰ or anyintegral number therebetween. Even larger libraries can be produced,especially where small cDNA fragments containing a few epitopes each areprepared, including up to 10¹¹ to 10¹² members in phage and 10¹² to 10¹⁵members in polysomes, or any integral number therebetween.

[0102] C. Detection and Target Protein Identification

[0103] 1. Labeled Target Proteins

[0104] One option is to label the proteins in the sample before theprotein sample is contacted with the package/antibody reagents. Reagentsthat are complexed to target proteins can then be detected from thesignal generated by the labeled target protein.

[0105] 2. Separation of Bound and Unbound Reagents

[0106] Certain analyses are conducted by contacting a sample potentiallycontaining target proteins with a population of package/antibodyreagents under conditions such that reagents specifically bind to targetproteins having an epitope that is recognized by the captured antibodyof the reagent. Unbound reagents are then selectively separated fromtarget protein/reagent complexes. Reagents that have formed complexeswith the target can then be collected, nucleic acids removed from thepackages, and the nucleic acid segments sequenced to determine theidentity of an epitope one of the target polypeptides. Often suchseparations are performed by attaching proteins within the sample to asupport, contacting the immobilized sample with the reagents and thenwashing away unbound reagents.

[0107] 3. Detection Reagents

[0108] Other detection approaches utilize various labeled detectionreagents that have specific affinity for a particular target protein.For instance, labeled antibodies that specifically bind to certaintarget proteins can be utilized to detect proteins bound topackage/antibody reagents. In a modified version of this approach,unlabeled antibodies that specifically bind to target proteins are usedand detection is accomplished using labeled antibodies that specificallybind to the unlabeled antibodies that have formed a complex with atarget protein. A potential problem associated with either of these twoapproaches is cross-reactivity of the detection reagent with thepolypeptide displayed by the package of the package/antibody reagent.However, the antibodies are expected to preferentially bind to thecaptured polypeptide rather than the displayed polypeptide because ofthe relative inaccessibility of the displayed polypeptide (sandwichedbetween a package and capture antibody) as compared to the capturedpolypeptide (bound only to the captured antibody).

[0109] Alternatively, the detection reagent can be composed of areplicable genetic package that displays a polypeptide at its surface, adetection antibody that specifically binds to the display polypeptide,and a label. The package of a detection reagent displays one of the samepolypeptides as displayed by one of the packages of the package/antibodyreagents brought into contact with the protein sample. Thus, the packageof the detection reagent includes a copy of the same heterologousnucleic acid segment as one of the package/antibody reagents (i.e., thesegment that encodes the polypeptide displayed by the two types ofreagents). Thus, package/antibody reagents and detection reagentssharing this feature, bear captured antibodies and detection antibodies,respectively, that have specific binding affinity for the same targetpolypeptide. A component of the detection reagent is labeled tofacilitate detection. Typically, the package is labeled to minimize thepossibility that the label interferes with the ability of the detectionantibody to bind to a target protein. The problem of cross-reactivitydescribed supra is lessened with this type of detection reagent becausethe bulk of these reagents impede their ability to gain access to thepolypeptide displayed by one of the package/antibody reagents.Consequently, these reagents offer better selectivity than the foregoingdetection reagents.

[0110] In use, a detection reagent bearing a detection antibody thatrecognizes an epitope on a target protein captured by one of thepackage/antibody reagents, forms a complex that contains: (i) thepackage/antibody reagent, (ii) the captured protein, and (iii) thedetection reagent. Because the package of the package/antibody reagentand the detection reagent both include the same nucleic acid segment,the sequence of the segment from either package can be sequenced todetermine an amino acid sequence that includes an epitope on the targetpolypeptide.

[0111] The second phage complex (i.e., the complex of the detectionreagent) can be a different phage. Alternatively, detection can beaccomplished with a “fusion protein captured” antibody display reagent.An example is a GST-cDNA expression product fusion, mixed with anantibody collection to capture one binding site of an antibody. Thisreagent, while not a replicable genetic package useful for identifyingthe target, can serve as a detection reagent for locating the target inan array, for example.

[0112] Given the similarity in composition, the preparation of suchdetection reagents closely parallels the preparation of thepackage/antibody reagents. For example, one typically prepares detectionreagents of this type by initially preparing a cDNA display library anda population of antibodies according to the methods described insections III.B.1 and 2. Then, usually prior to contacting members of thedisplay library with the antibody population, members of the displaylibrary are labeled. In certain instances, this is accomplished bylabeling the packages of the display library either by covalentattachment or using labeled antibody that binds to the package (i.e.,labeled anti-package antibody). A fluorescent label is often utilizedbut other detectable labels can be used as well (see the detectionsection infra). The members of the labeled display library are thenincubated with the antibody population to form a collection of detectionreagents. The resulting detection reagents are essentially identical tothe package/antibody reagents described above, except that the detectionreagents bear a label whereas the package/antibody reagents do not.

[0113] However, as noted above, a fusion protein captured antibodydisplay reagent can also be utilized as a detection reagent. Thus, aGST-cDNA expression product fusion mixed with an antibody collection tocapture one binding site of an antibody is one specific example. Suchreagents can be useful in locating a target in an array for example.

[0114] Even though the capture antibody of the package/antibody reagentand the detection antibody of the detection reagent recognize the sametarget protein, both the package/antibody reagent and the detectionreagent can bind to the same copy of the target protein because the twotypes of antibodies recognize different epitopes on the same protein.Nonetheless, as noted above, package/antibody reagents and detectionreagents that bind to the same target protein share a common nucleicacid segment (namely, the segment that encodes for the same polypeptidedisplayed on both types of reagents). Consequently, the amino acidsequence of an epitope of the target can be determined by sequencing thenucleic acid segment from either the package/antibody reagent or thedetection reagent.

[0115] 4. Identification

[0116] Once package/antibody reagents that have formed complexes with atarget protein have been detected, the captured target protein can beidentified. Generally, this involves removing the nucleic acid, or atleast the segment encoding the displayed polypeptide, from the packageof a package/antibody reagent. The nucleic acid or segment is thenusually amplified using known techniques and then sequenced, typicallywith commercial sequencing instruments.

[0117] D. Applications

[0118] The individual package/antibody reagents or libraries ofpackage/antibody reagents can be utilized to detect the presence of arelatively small number or very large number of target proteins indiverse sample types. The reagents can be used to probeprotein-containing solutions or other types of matrices that includeproteins. As indicated in the Background section, a common practice inproteomics applications is to at least partially resolve complex proteinmixtures into component proteins by gel electrophoresis, especiallymultiple dimensional electrophoresis (e.g., two-dimensionalelectrophoresis). The package/antibody libraries disclosed herein can beutilized to detect and identify the proteins in the different locationson the gel.

[0119] For example, using the methods described above, one can prepare alibrary of package/antibody reagents that have specific binding affinityto the proteins within a cell or tissue under investigation. The cellsor tissue used to prepare such a library are also processed to obtain atotal protein fraction that includes all of the expressed proteins inthe cell or tissue of interest (of course, a subset of such proteinssuch as from a particular subcellular compartment can also be probed).

[0120] The proteins in this fraction are applied to a gel and separatedinto many different spots (often thousands) that is typical oftwo-dimensional electrophoretic formats. Various electrophoretic methodscan be used in combination to achieve separation. Examples of suchmethods include, but are not limited to, zone electrophoresis(separation of proteins on the basis of their intrinsic charge-to-massratio), isoelectric focusing electrophoresis (proteins separatedaccording to their isoelectric points) and gel electrophoresis methodsthat separate on the basis of size. Such methods are discussed, forexample, by Hochstrasser, D. F., et al. (1988) Anal. Biochem. 173:424;O'Farrell, P. H. (1975) J. Biol. Chem., 250:4007; and Anderson, N. G.and Anderson, N. L. (1996) Electrophoresis 17:443. In many applications,proteins are separated in one dimension and then the gel partiallyrotated to achieve further electrophoretic separation of the proteins.For example, certain protein separation procedures involve iso-electricfocusing along a first dimension followed by SDS-PAGE electrophoresisalong a second dimension. See, e.g., Hames et al, 1990, GelElectrophoresis of Proteins: A Practical Approach, IRL Press, New York;Shevchenko et al., 1996, Proc. Natl. Acad. Sci. USA 93:1440-1445;Sagliocco et al., 1996, Yeast 12:1519-1533; Lander, 1996, Science274:536-539.

[0121] The proteins in each spot can be rapidly detected and identifiedwith the library of reagents prepared from the cells or tissue. Onedetection option involves excising individual spots (or a small numberof spots) from the gel, eluting the proteins within the spot(s) from theexcised section and subsequently transferring the eluted protein(s) ontosome type of support. A variety of different supports can be utilizedincluding, but not limited to, glass, cellulose sheets, and variousmembranes (e.g., nylon, and polyvinylidene difluoride (PVDF)). Theimmobilized protein is then “stained” by incubation with thepackage/antibody library, followed by washing to remove unbound andnon-specifically-bound package/antibody reagents. Packages that remainbound to the support are recovered by elution with an appropriatesolution. One suitable wash solution is one of low pH. Filamentous phageare stable to pH 2.2 (glycine buffer) for 10 minutes at roomtemperature. T7 phage are less stable to low pH so an elution buffer of1%SDS in PBS is generally used. The nucleic acids of those replicablegenetic packages eluted from the support are subsequently amplified andthe nucleic acid (or segment thereof) that encodes the displayedpolypeptide is sequenced. Translation of this nucleic acid sequence toan amino acid sequence provides a portion of the sequence of theimmobilized protein, thus identifying the protein(s) present in thesection of the gel that was removed.

[0122] Other analyses involve the in situ identification of many (orall) proteins in a two-dimensional gel. This is accomplished byincubating the gel or a replica blot thereof with the package/antibodylibrary. Unbound and non-specifically bound package/antibody reagentsare washed away, thus leaving an array of immobilized proteins complexedwith package/antibody reagents (see FIG. 15A). Reagents that remainbound to the gel are picked (eluted) from different spots on the gel,amplified and then sequenced.

[0123] In certain gel analyses such as just described, several or manydifferent reagents can be isolated from any given spot on the gel. Thisis a consequence of the fact that each package/antibody reagent cancontain a different segment of the cognate full-length nucleic acidclone, each segment expressing one or a few (or in some longer clones,many) different epitopes. Thus, in certain instances (depending, forexample, on the method of cDNA preparation) the entire full-length cDNAsequence can be deduced from the nested sequences of several of therecovered reagents. Such methods then become an additional tool fordetermining the sequence of a given expressed genome. In those caseswhere the proteins under analysis derive from organisms with fullysequenced genomes, even a short segment from a single package clone canserve to unambiguously identify the protein(s) in a spot from a 2-D gel.

[0124] Even though proteins have the same primary sequence theysometimes can appear at different locations on a gel because ofdifferential processing that results in proteins being differentiallymodified. For example, proteins of the same sequence that aredifferentially phosphorylated or glycosylated may become separated fromone another on the gel. The antibody/package reagents described hereincan be utilized to detect the presence of such differentially modifiedproteins. More specifically, different spots on a gel that include thesame proteins can be identified by determining which spots containproteins that bind to the same package/antibody reagents.

[0125] Such analyses can be conducted without sequencing, thus allowingone to rapidly identify spots that have differentially modified proteinsthat have the same primary sequence. In general, this can be done bylysing the packages of the reagents that have formed a complex withproteins in the spots to expose the heterologous nucleic acid and thenprobing the gel with a labeled nucleic acid probe that is complementaryto at least a segment of the heterologous nucleic acid under conditionssuch that the probe can hybridize with a complementary segment.

[0126] One approach for conducting such analyses that simplifies thehybridization step is to utilize isothermal tags. Isothermal tags referto nucleic acid sequences that have the same base composition but whichdiffer in the ordering of the bases. Because they have the same overallbase composition, isothermal tags have the same melting temperature.Consequently, one can conduct hybridization and washing steps withmultiple probes under a single set of conditions, thereby significantlysimplifying the screening process. The isothermal tags are introduced byattaching the tag to the heterologous nucleic acid sequence that encodesthe expressed polypeptide prior to incorporating the heterologoussequence into the replicable genetic package.

[0127] IV. Arrays of Package/Antibody Reagents and Methods of Use

[0128] A. General

[0129] The replicable genetic package/antibody reagents described insection III can also be utilized in a variety of array formats. Certainarrays include package/antibody reagents that are immobilized on asupport, and thus constitute an array of immobilized antibodies that cancapture a target protein for which the antibodies have specific bindingaffinity (see, e.g., FIG. 15B). With this type of array, the identity ofa target protein that is complexed to a reagent on the array can bedetermined from the sequence of the segment of the heterologous nucleicacid of the reagent that has captured the target protein. As pointed outabove, this is because the sequence of the segment encodes for anepitope that is shared by the polypeptide expressed by the reagent andthe captured target protein.

[0130] Arrays of this type can be utilized in a number of differentapplications similar to the profiling applications conducted usingnucleic acid arrays. Thus, the arrays can be utilized to detectqualitatively or quantitatively the presence of one or more proteins forcells or tissues under a particular set of conditions or stage ofdevelopment. One can also conduct differential expression studies inwhich proteins expressed under one set of conditions are compared toproteins expressed under another set of conditions. Because the arraysdisclosed herein monitor some or all of the proteins in a cell or tissueinstead of mRNA levels as do nucleic acid arrays, the arrays providedherein can yield a more accurate view of the actual components thatregulate biological process. Knowledge of the identity and/or amount ofeach protein enables one to gain a more complete understanding ofbiological processes.

[0131] Other arrays that are related to those exemplified in FIG. 15Balso include proteins captured by the antibodies (see, e.g., FIG. 15C).Arrays of this type can display functional polypeptides and can beutilized in a variety of studies on protein/protein interactions.

[0132] B. Array Preparation/Structure

[0133] 1. Immobilization of Replicable Genetic Packages

[0134] The components of the package/antibody reagents that areimmobilized on a support to form the array can be prepared according tothe preparation methods described in section III. If not alreadyavailable, the cDNA display library and the population of captureantibodies are prepared. Certain arrays are then formed by immobilizingmembers of the cDNA display library onto locations on a substrate. ThecDNA display library can be immobilized in a number of ways. Onetechnique is to spot aliquots of package clones in specific locations onthe support such that different clones are spatially addressable. Thealiquots can be spotted utilizing a variety of available techniques suchas using modified ink jet printers, robotic spotters and capillaryarrangements. Arrays of a variety of densities can be prepared utilizingsuch approaches. Certain arrays have densities of at least 10, 10², 10³,10⁴, 10⁵, 10⁶, 10⁷ or 10⁸ locations/cm² or any integral number oflocations therebetween.

[0135] Other methods can be utilized to immobilize reagents when thereplicable genetic packages are filamentous and lytic phage. Forinstance, if fd phage are utilized, an array of polypeptide-displayingphage can be created by growing bacterial micro-colonies expressingdisplay phage (i.e., bacteriophage with the display polypeptide on thesurface). If lytic phage (e.g., T7) are used, then a lawn ofmicro-plaques is grown. The array of colonies/plaques is then replicatedonto a support, followed by treatment to achieve stable attachment ofthe phage to the support surface using standard methods of colony andplaque lifts as described in Sambrook, et al. (1989) Molecular Cloning:A Laboratory Approach, 2nd ed., Cold Spring Harbor Laboratory Press.Such methods using colonies or plaques do not require a uniform array(i.e., deposition of package clones at particular locations), but can beconveniently prepared by simply plating or spreading the phage on agrowth medium and permitting the phage to replicate prior to replicationand immobilization of the colonies or plaques on the support. Thus, suchapproaches avoid the need for automated arraying equipment.

[0136] These methods of generating either micro-colonies ormicro-plaques can be utilized to generate high density protein arraysthat otherwise would be difficult to prepare utilizing standarddeposition methods. High densities of proteins can be achieved bypreparing arrays in this fashion because very small colonies or plaques(e.g., approximately 5 microns or less in size) can be formed. Ingeneral densities of at least 10² to at least 10⁸ locations/cm² areobtained. Thus, utilizing such techniques arrays having at least 10 ⁵,10⁶, 10⁷ or 10⁸ locations/cm² or any integral number therebetween can beprepared. In some instances, even higher density arrays having 10⁹ or10¹⁰ locations/cm² or any integral number therebetween can be prepared.

[0137] Regardless of the particular method by which the display libraryis attached to the support, usually a replica of the resulting array ismade and preserved to provide an archival collection of the packageclones in which the location of the various clones are maintained. Thus,at this stage one has a high density array of package clones, eachexpressing a polypeptide (of fragment thereof) from the cells or tissuebeing studied. This array of displayed proteins can be utilized directlyin various applications as described further infra.

[0138] 2. Complexing Antibodies to Immobilized Packages

[0139] Once the array of display packages has been formed, it iscontacted with an antibody collection, with antibodies forming complexesat those locations that contain immobilized reagents that displaypolypeptides to which the antibodies have specific binding affinity. Asdescribed in section III.B.3, because the density of the polypeptidedisplayed on package is designed to be relatively low, multivalentantibodies binding to a displayed protein typically have one or moreadditional sites that are available to bind with target protein in asample. Consequently, free antibody recognition sites are displayed ateach location of the array. The specificity of the antibodies within thelocation corresponds to the epitope of the polypeptide displayed by thepackage immobilized at that location. Hence, at this step of theprocess, the array is effectively an anti-target protein antibody array,and it can be utilized to evaluate the global protein complement (orsome subset thereof) of the cells of interest.

[0140] One alternative to the foregoing methods is to generate thepackage/antibody reagents and then attach these to the support. Thereagents can then be deposited using the modified ink jet printers,robotic spotters and capillary arrangements described supra, forexample.

[0141] 3. Array Structure

[0142] Hence, utilizing methods such as those just described, a widevariety of arrays in which polypeptides are immobilized at differentlocations on a substrate can be formed. The arrays can differ in anumber of aspects, including for example, the type of polypeptideavailable for binding, the density of locations (and thus polypeptides)on the array, and the number of different polypeptides immobilized tothe array.

[0143] For example, certain arrays contain immobilized package/antibodyreagents, thus forming an array of displayed antibodies (see FIG. 15B).The antibodies displayed in these arrays can be of a number of differenttypes. Suitable antibodies in such arrays include, but are not limitedto, diabodies, tribodies, tetrabodies, IgG antibodies, and can also bethe antibody from another package/antibody complex, provided theantibody of the complex is multivalent.

[0144] Other related arrays also include immobilized package/antibodyreagents, but also include polypeptides that have been captured by thecaptured antibody of the immobilized package/antibody complexes (see,e.g., FIG. 15C). In such arrays, the protein captured by the antibodycan be of a number of different types. The captured protein in certainarrays is a functional protein. In general, functional proteins refer toproteins that retain the activity or a substantial portion of theactivity associated with the particular protein. Functional proteins canbe obtained and isolated from a cell. These proteins typically includethe post-translational modifications associated with the protein asobtained from a cell, are full-length or substantially full length, andproperly folded such that the protein is functional.

[0145] The density of locations on the substrate can include any of thedensities listed above. Typically, polypeptides within a location haveare the same (e.g., have the same amino acid sequence) but this is notrequired. Similarly, at least some, and in other instances many, of thedifferent locations on the support have different polypeptidesimmobilized therein. However, with some arrays at least some of thelocations will have the same polypeptide immobilized therein. As usedherein, “different” polypeptides refer to polypeptides that havedifferent amino acid sequences and/or that differ in some othercharacteristic or property (i.e., proteins with a “differentialmodification”). The characteristic or property differentiating thepolypeptides often is a difference in postranslational modification,such as glycosylation, acetylation or post translational processingresulting from protease activity, for example. Polypeptides referred toas being the same are those having the same amino acid sequence, and canalso refer to polypeptides that additionally have the samepostranslational modifications. The number of different polypeptidesthat are immobilized on the array can be less than 10, but moretypically is at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100different polypeptides or any integral number therebetween. Certainarrays have even more immobilized polypeptides, such as at least 10²,10³, 10⁴ or 10⁵ different polypeptides or any integral numbertherebetween.

[0146] The locations on the substrate to which members are immobilizedcan vary significantly in size and can be of different shapes. Thesubstrate can be essentially any material to which the cDNA library canbe attached and which is physically and chemically compatible with washsolutions that are used to remove unbound or non-specifically boundproteins from the array. Suitable supports include, but are not limitedto, polymeric materials such as plastics, resins, cellulose, nylon, PVDFand polysaccharides, as well as silica or silica-based materials andinorganic glasses. The substrates can be in a variety of forms such assheets, membranes, tubes and beads, for example.

[0147] C. Contacting Array with Protein Sample

[0148] When a protein-containing sample is applied to the array,proteins become complexed or captured at those sites that containantibodies that recognize the various epitopes on the proteins. Thesesites are, in turn, the sites at which the cDNAs encoding the capturedproteins also reside. Depending upon the distribution of thepackage/antibody complexes on the array, in some instances a particularprotein can be captured at a number of different sites on the array.This feature, however, can be controlled and can be used to advantage asdescribed infra. After proteins in solution have had sufficient time toform complexes with the immobilized package/antibody reagents, the arrayis optionally washed with a rinse solution to remove unbound and/ornon-specifically bound protein.

[0149] D. Detection and Identification

[0150] A variety of different options for detecting which locations ofthe array have captured protein from the sample are availableand-generally parallel those methods listed in section III C. In brief,one option is to label the proteins within the sample prior to applyingthe sample to the array. Once the labeled proteins have had anopportunity to form complexes with the immobilized reagents on thearray, unbound or non-specifically bound labeled proteins can optionallybe washed away during a washing step. Target proteins that remain boundto the array can subsequently be detected.

[0151] Alternatively, detection reagents such as those described insection III.C.3. can be washed over the array. Those detection reagentsthat specifically bind to target proteins captured on the array bind tothe array to form a complex that includes: (i) immobilized reagent, (ii)captured target protein, and (iii) detection reagent. An optionalwashing step can be performed to remove unbound or non-specificallybound detection reagent prior to detecting complexes. Regardless of thedetection reagent utilized, methods can be conducted in a manner suchthat the extent of binding of the labeled detection reagent to eachlocation on the array is a reflection of the relative amount of proteinbound to any particular spot. Thus, certain methods can be utilized in aquantitative fashion to obtain quantitative information on differentproteins within a sample. The goal with other methods is to obtainqualitative information. For instance, one can use protein arrays of thetype described herein to obtain a “fingerprint” of the protein contentof a cell or tissue under particular growth conditions, at a particularstage of development or differentiation, or after exposure to aparticular environmental stimulus.

[0152] In certain comparative methods, one is primarily interested inthe protein expression pattern observed on the array and it is notnecessary to determine the identity of each target protein that is boundto the array. For instance, the expression pattern can be used to trackchanges in cells at different periods (e.g., different developmentalstages) or under different conditions. With the arrays provided herein,such generalized information can be obtained for some, many, or all ofthe proteins within a cell or tissue.

[0153] However, considerably more information can be obtained,information that is not readily obtainable using conventional antibodyarrays. In particular, the methods disclosed herein can be used torapidly identify the protein captured at every (or any) location on thearray, even if the protein is completely novel. This capability ispossible with the package/antibody arrays provided herein because eachsite of the array physically contains the cDNA sequence of the proteinunder evaluation at that site (i.e., of the protein captured at thatsite). To interrogate the actual sequence and thus identity of anyparticular captured protein, it is only necessary to establish thesequence of a segment of the nucleic acid of the package at the locationat which the protein is captured. The sequence of the nucleic acidsegment includes the sequence of a shared epitope on the capturedpolypeptide.

[0154] The sequence of the nucleic acid segment that corresponds to ashared epitope of a captured protein can be determined in various ways.One approach utilizes the replica of the array that preserves thelocation of the various package/antibody reagents on the array. Inparticular, a package from the archival replica is removed from thelocation on the replica that corresponds to the location on the array atwhich a target protein is bound. The nucleic acid from the removedpackage is extracted and sequenced to reveal the primary nucleic acidsequence; this sequence of course can be utilized to determine a primaryamino acid sequence that includes the sequence of an epitope that isshared by the polypeptide displayed by the reagent and the capturedprotein. Thus, for example, if a set of sites on the array are seen todiffer in their protein content, the DNA from packages from the replica(master archive) corresponding to those sites can be sequenced to obtainthe primary amino acid sequence of at least a portion of the protein(s)bound at those sites.

[0155] The identification step can be accomplished without the use of anarchival replica by using detection reagents (see section III.3.C.).Since the polypeptide displayed by the package of the display reagentthat binds to the target protein is the same as the captured protein andthe polypeptide displayed by the immobilized package/antibody complex,the segment of the nucleic acid that encodes the polypeptide of thedetection reagent and on the immobilized reagent are the same. Thus, onecan determine the sequence of the segment of an immobilized reagent thathas captured a protein from the sequence of the nucleic acid of thedetection reagent complexed to the captured protein.

[0156] The detection reagent bound to any site of interest can beremoved using microdissection techniques and the segment of theheterologous nucleic acid of the detection reagent sequenced todetermine the corresponding sequence in the immobilized reagent thatcaptured the protein. Suitable microdissection instruments include acapture microdissection instrument. In some instances, these instrumentsare lasers such as those marketed by Arcturus Engineering of MountainView, Calif. Once a package is removed, the nucleic acid is removed,typically amplified using established methods and then sequenced. Suchtechniques can be used for locations on the array that are as small as10 microns in diameter.

[0157] E. Optional Normalization Techniques

[0158] As indicated above, some sites in the array may includepackage/antibody reagents that display the same polypeptide. Hence, suchsites will capture the same specific antibodies, and thus capture thesame proteins from the applied sample. This is especially true ofproteins that are highly transcribed within the cells or tissues beingstudied. Normalization procedures known in the art can be utilized toaddress this issue when preparing the cDNA libraries for thisapplication (see, e.g., Sambrook, et al. (1989) Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, ColdSpring Laboratory, N.Y.).

[0159] However, even with normalization, in some instances methods forarraying the members of the cDNA display library result in many of theexpressed polypeptides being positioned at multiple sites. Such arrays,however, can be used to advantage. For example, when several sitesexpress a similar segment of a particular cDNA (i.e., the displayedpolypeptide fragment borne by packages contains the same epitopes andthus captures the same set of antibodies), the intensity of the signalfrom those sites will rise and fall in concert with changes in the levelof protein binding to those sites. Thus, such sites can serve asinternal controls in verifying the changes observed at other sites.

[0160] The presence on an array of multiple sites having reagents thatdisplay related proteins can also be used to good effect indistinguishing between modified and unmodified proteins. For example,reagents displaying polypeptides that contain different segments of thesame protein (and therefore contain different sets of epitopes) may bepresent at several different locations on the array. In some instances,the polypeptide expressed by the immobilized reagent contains only asingle epitope that can be modified by phosphorylation or glycosylation,for example. Such modification may allow or prevent the binding of thecognate antibodies, thereby allowing differentially modified proteins atthe different locations to be distinguished. This feature can provideimportant insight into regulatory mechanisms, for example.

[0161] V. Target Protein Identification

[0162] A. Detection Options

[0163] As set forth above, depending on the nature of the detectiontechnique utilized, labeled target protein or various labeled detectionreagents (e.g., labeled antibodies or labeled packages) can be used todetect the formation of a complex between a target protein andpackage/antibody reagent. A variety of different labels can be utilizedin these detection schemes. The proteins, antibodies or packages can belabeled with any of a variety of different types of labels, provided thelabel does not interfere with the formation of a complex between apackage/antibody reagent and a target protein and can generates adetectable signal once such a complex is formed. Suitable labelsinclude, but are not limited to, radiolabels, chromophores,fluorophores, electron dense agents, NMR spin labels, a chemical tagsuitable for detection in a mass spectrometer, agents detectable byinfrared spectroscopy or NMR spectroscopy, and enzyme substrates orcofactors for example. Radiolabels, particularly for spatially resolvedproteins, can be detected using phosphor imagers and photochemicaltechniques.

[0164] Certain methods utilize fluorophores since various commercialdetectors for detecting fluorescence from labeled proteins areavailable. A variety of fluorescent molecules can be used as labelsincluding, for example, fluorescein and fluorescein derivatives,rhodamine and rhodamine derivatives, naphythylamine and naphthylaminederivatives, benzamidizoles, ethidiums, propidiums, anthracyclines,mithramycins, acridines, actinomycins, merocyanines, coumarins, pyrenes,chrysenes, stilbenes, anthracenes, naphthalenes, salicyclic acids,benz-2-oxa-1-diazoles (also called benzofurazans), fluorescamines andBodipy dyes.

[0165] B. Sequence Determination

[0166] Once a complex has been detected, often the methods next involvedetermining the sequence of the nucleic acid of the package of apackage/antibody reagent to which a target protein is bound. Bytranslating the nucleic acid sequence, one can determine an amino acidsequence that includes the epitope of the target protein. In view of thedetection options described supra for both array and non-array formats,it should be understood that the sequence of a segment of theheterologous nucleic acid of a reagent that complexes with targetprotein can be determined in a number of different ways. One option issimply to isolate the reagent, extract the nucleic acid segment andsequence it. The sequence can also be determined by extracting thesegment and utilizing nucleic acid probes to detect the presence ofcomplementary segments in nucleic acids that have been extracted fromreagents that have captured target protein. Alternatively, the sequencecan be deduced from the sequence of the heterologous nucleic acid of adetection reagent that complexes with the target protein captured by areagent.

[0167] Generally, the nucleic acid is amplified before sequencing iscommenced. Amplification is typically conducted using the polymerasechain reaction (PCR) according to known procedures. See generally, PCRTechnology: Principles and Applications for DNA Amplification (H. A.Erlich, Ed.) Freeman Press, NY, N.Y. (1992); PCR Protocols: A Guide toMethods and Applications (Innis, et al., Eds.) Academic Press, SanDiego, Calif. (1990); Mattila et al., Nucleic Acids Res. 19: 4967(1991); Eckert et al., PCR Methods and Applications 1: 17 (1991); PCR(McPherson et al. Ed.), IRL Press, Oxford; and U.S. Pat. Nos. 4,683,202and 4,683,195. Other suitable amplification methods include the ligasechain reaction (LCR) (see, e.g., Wu and Wallace, Genomics 4:560 (1989)and Landegren et al., Science 241:1077 (1988); transcriptionamplification (see, e.g., Kwoh et al., Proc. Natl. Acad. Sci. USA86:1173 (1989)); self-sustained sequence replication (see, e.g.,Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990)); andnucleic acid based sequence amplification (NABSA) (see, e.g., Sooknanan,R. and Malek, L., Bio Technology 13: 563-65 (1995)).

[0168] Sequencing of the amplified sequence can be conducted with any ofa number of different commercially-available nucleic acid sequencers.

[0169] VI. Samples

[0170] The methods disclosed herein can be used with a wide range ofprotein samples types, provided the protein sample can be brought intocontact with the package/antibody reagents (or arrays containing suchreagents) such that complexes between target proteins in the sample andthe package/antibody reagents can form. The samples can contain arelatively small number of proteins or can contain a large number ofproteins, such as all the proteins expressed within a cell or tissuesample, for example. Samples can also contain a subset of the proteinsof a cell or tissue, such as the proteins within a particular cellularorganelle or subcellular compartment.

[0171] Samples can be obtained from any organism or can be mixtures ofsynthetically prepared proteins or combinations thereof. Thus, suitablesamples can be obtained, for example, from microorganisms (e.g.,viruses, bacteria and fungi), animals (e.g., cows, pigs, horses, sheep,dogs and cats), hominoids (e.g., humans, chimpanzees, and monkeys) andplants. The term “subject” as used to define the source of a sampleincludes all of the foregoing sources, for example. The term “patient”refers to both human and veterinary subjects. The samples can come fromtissues or tissue homogenates or fluids of an organism and cells or cellcultures. Thus, for example, samples can be obtained from whole blood,serum, semen, saliva, tears, urine, fecal material, sweat, buccal, skin,spinal fluid, tissue biopsy or necropsy and hair. Samples can also bederived from ex vivo cell cultures, including the growth medium,recombinant cells and cell components. In comparative studies toidentify potential drug or drug targets (see infra), one sample can beobtained from diseased cells and another sample from non-diseased cells,for example.

[0172] If the sample contains cellular debris or other non-proteinmaterial that might interfere with the analysis, such materials can beremoved using any of a variety of known separation techniques including,for example, forcibly exuding the sample through sieve material,filtration and centrifugation.

[0173] VII. Exemplary Utilities

[0174] A. General

[0175] The methods, reagents and arrays described herein can be utilizedin a variety of proteomic applications. In general, the methods andcompositions can be utilized to detect, characterize and/or identifymany proteins (e.g., tens, hundreds or thousands of proteins in someinstances). With such capabilities, the methods and compositions haveutility in a wide range of applications including, but not limited to:(i) various analytical applications (e.g., monitoring certain proteinlevels as a function of external stimuli, or detecting specific proteinsin complex compositions for identification purposes); (ii) clinicalapplications (e.g., detecting and/or monitoring compositions of normaland diseased cells and tissues, diagnosing or monitoring disease,testing drug candidates for therapeutic efficacy, and toxicity testing);and (iii) molecular biology and genetic research (e.g., characterizingor monitoring molecular expression levels of gene products anddetermining the effects of the addition, mutation, deletion ortruncation of a particular gene).

[0176] More particularly, the methods and compositions can be utilizedto identify the primary sequence of any, most, or all of the proteins intissues, cells or subcellular compartments (e.g., such as at least 20%,30%, 40%, 50%, 60%, 70%, 80% or 90% of the proteins in the tissue, cellor compartment). The methods and compositions can also be used for thesame purpose in the analysis of complex protein mixtures that have beenseparated in one- or two-dimensional separation formats (e.g., one- ortwo-dimensional electrophoretic gels), or similar analyses conducted inarray-based formats (including arrays that include unknown proteins andarrays in which known proteins are immobilized at unknown locations).

[0177] A. Screening

[0178] One use of the methods and compositions is to examine eitherqualitatively or quantitatively the proteins that are expressed in aparticular tissue or cell. However, one can also screen a large numberof samples to detect the presence of individual proteins or smallsubsets of proteins. The specific reagents or panels of reagentsdescribed herein make such analyses facile. Screens of this type findutility in a variety of medical applications as described further below.Such screening methods can also be utilized in various identificationapplications, such as identifying a particular cell type, species oreven an individual of a species.

[0179] B. Reagents and Arrays for Specific Analyses

[0180] The reagents provided can be tailored to any of a number ofparticular applications. For example, individual package/antibodyreagents that have specific binding affinity to any protein of interestcan be created, provided at least some portion of the primary sequenceof the protein is known, using the methods and compositions describedherein. This is done by: (i) cloning the cDNA sequence of the targetprotein into a replicable genetic package to obtain a package displayingthe protein of interest (or cloning a small population of fragments toobtain a small library of display packages); (ii) exposing the displaypackage to an antibody collection to capture the specifically reactiveantibodies; and (iii) recovering the resulting package/antibody reagent.The resulting reagent can be used in the detection of the protein ofinterest in a protein sample. Of course a collection of such reagentscan be prepared to detect a particular group of proteins of interest.

[0181] Domain-specific or cell fraction-specific populations ofantibodies can be readily produced in a related manner. Such methodsgenerally involve: (i) creating a library of package/antibody reagentlibraries that represent the total protein from a cell as describedherein; (ii) enriching for those package/antibody reagents that arespecific to the cell location or fraction of interest (e.g., bycontacting the reagents to the apical domain of an epithelial cell toenrich for reagents that specifically bind to proteins in thisparticular location); and (iii) recovering and amplifying such packagesfor use as an immunogen in one of the antibody preparation formatsdescribed in section III.B.2. The resulting antibodies can be utilizedin a variety of applications to detect proteins from the particulardomain or cell fraction of interest.

[0182] Arrays of full-length functional proteins such as discussed abovecan be constructed by: (i) preparing a cDNA display library as describedin section III.B.1.; (ii) arraying the members of the cDNA displaylibrary onto a support; (iii) exposing the array to an antibodypreparation prepared using a corresponding display library as theimmunogen as disclosed in section III.B.2. and washing away the unboundantibodies; and (iv) contacting the array with a protein preparationfrom a cell or tissue of interest, thereby capturing specific proteinsin specific locations on the array (see FIG. 15C). Thus, arrays preparedaccording to steps (i)-(iii) can be utilized to capture functionalproteins which can be assayed in situ for their activity (e.g., enzymeactivity) and to detect other proteins in a mixture that interact withthe functional proteins displayed in the array.

[0183] Hence, a variety of protein/protein interactions can be probedusing reagents having the general structure: package/displayedprotein/captured antibody/captured functional protein. By using labeledtarget proteins, for example, reagents of this type can be used todetect target proteins in a sample that interact with the functionalprotein. The sequence of the package that is complexed with a targetprotein identifies the functional protein that interacts with the targetprotein. The ability to conduct screens with functional proteins ascompared to conventional phage display libraries means that one canconduct assays with full length proteins that have been modified astypically occurs within a cell and that are properly folded. Thepolypeptides in conventional polypeptide display libraries, in contrast,are often fragments, have not been modified and may not be properlyfolded.

[0184] C. Comparative Analyses

[0185] Diverse comparative studies can be performed with the methods andcompositions provided herein. By comparing qualitatively and/orquantitatively the complement of proteins (or some subset thereof)expressed in cells or tissues from multiple different samples, one canidentify a particular protein or group of proteins that aredifferentially expressed between the samples. A wide variety of suchcomparisons can be made. For example, one can conduct comparisons ofprotein expression in cells or tissues exposed to different conditionsor at different stages of development or differentiation, for example. Aprotein whose expression level varies between the different samples canbe considered a “marker” or a “fingerprint” protein. Thus, dependingupon the nature of the comparison being conducted, one can identifyindividual markers or fingerprint proteins that correlate with aparticular cellular state, stage of development or stage ofdifferentiation, for example. As described more fully infra, marker orfingerprint proteins can be utilized in the development of a widevariety of different screening and diagnostic methods.

[0186] Expression levels for combinations of differentially expressedproteins can be used to develop a “fingerprint” or an “expressionprofile” that is characteristic of a particular cellular state, stage ofdevelopment, stage of differentiation, or cell or tissue type, forexample. Expression profiles or protein fingerprints contain a pluralityof differentially expressed proteins, usually at least 2, 3, 4, 5, 6, 7,8, or 9 proteins. Other profiles or finger prints include considerablymore differentially expressed proteins such as 10, 20, 30, 40, or 50 ormore proteins, or any integral number of proteins therebetween. Stillother profiles can include several hundred proteins. Certain profilesinclude all of the proteins know to be correlated with a particularcellular state, stage of development or type of cell, for example. Insome instances, the term “fingerprint” can be used in a general sense torefer to the particular pattern of expression observed on an array.

[0187] One example of a comparative analysis is an analysis in which oneidentifies a subset of proteins that are expressed in common bydifferent cells or tissues. One approach for doing this involvespreparing a library of package/antibody reagents from tissue A (seesection III) and then using this library to probe a two-dimensional gelthat contains separated proteins from tissue B (see FIG. 15A). Anotheroption is to prepare a cDNA display library from nucleic acids isolatedfrom tissue A. This cDNA library is then used to capture antibodiesgenerated by immunizing an animal with proteins from tissue B to form alibrary of package/antibody reagents. The resulting reagent library canthen be utilized to probe tissue A, B or any other tissue C.

[0188] As a more specific illustration of the utility of suchcomparative studies, the following examples demonstrate how the methodsand compositions can be utilized to identify potential drug targetsand/or candidates and in toxicological investigations. For example, themethods can be utilized to identify proteins that are differentiallyexpressed in diseased cells as compared to normal cells. Suchdifferentially expressed proteins can serve as targets for drugs orserve as a potential therapeutics. In a related fashion, the methods canbe used in toxicology studies to identify proteins that aredifferentially expressed in response to particular toxicants. Suchdifferentially expressed proteins can serve as potential targets or aspotential antidotes for particular toxicants.

[0189] The comparative studies necessary to identify the differentiallyexpressed proteins often are conducted on an array because of the easeof detection and drawing comparisons between the expression patternobserved on the different arrays. The comparisons can be conducted invarious ways. One option is to separately apply different samples todifferent arrays such that the comparative analyses are conducted inparallel. Another option is to differentially label the differentprotein samples and then apply the samples to a single array. Proteinsfrom one sample can be differentiated from those from another sample onthe basis of the different labels. The ratio of the labels can beutilized to determine the ratio of the concentrations of the proteinsfrom the samples.

[0190] D. Diagnostic Applications

[0191] The results of comparative studies are transferable to a varietyof diagnostic applications. For example, “marker” or “fingerprint”proteins identified during comparative studies as being characteristicof a particular disease can be used to diagnosis individuals todetermine if they have the disease correlated with the marker. Thesemarkers can also be used in medical screening tests.

[0192] Furthermore, as described supra, once marker or fingerprintproteins have been identified, one can rapidly generate package/antibodyand/or antibody reagents that specifically recognize such proteins.These reagents can be used in the preparation of diagnostic kits anddevices. For example, through comparative analyses such as those justdescribed, one can identify diagnostic markers (e.g., cell surfaceantigens or serum proteins) for immunodiagnostic assays. Purified markeror display packages displaying the identified epitope can then beutilized to generate antibodies having specific binding affinity to theprotein marker. Such antibodies can be used in immunological stainingtechniques to localize the protein in diseased cells or to rapidlyscreen patients for the presence of the protein.

[0193] E. Creating Databases

[0194] The methods described herein can also be used to generateinformation on proteins that is used to populate a database. Anexemplary database might include, for example, the identity and quantityof protein present in cells or tissues under a particular set ofconditions. Other entries in the database could include similarinformation for cells or tissues under a variety of other conditions.Information in the databases can be further cross referenced with avariety of information regarding the source and identity of the sample,method of sample preparation and the like.

[0195] F. Establishing Structure Activity Relationships and MetabolicEngineering

[0196] The methods provided herein have further utility in conductingstructure activity studies. For instance, the methods can be used todetermine the effect that certain chemical agents or combination ofagents have on protein expression patterns. Alterations to the agent orcombination can then be made and protein expression reassessed todetermine what effect if any the alteration has on protein expression.Such studies can be useful, for example, in making derivatives of a leadcompound identified during initial drug screening trials.

[0197] Metabolic engineering studies can also be performed utilizing thepresent methods and compositions. In such studies, for example, a genecan be modified using established genetic engineering techniques or thepromoter for the gene is modified to increase or decrease the expressionlevel of the gene. The methods described herein can then be used todetermine what effect, if any, the genetically engineered changes haveon proteins within a cell harboring the changes other than on theprotein encoded by the genetically engineered gene.

[0198] G. Endocytosis and Transcytosis Assays

[0199] Active transport of compounds into or through cells can occur byvarying mechanisms including endocytosis and transcytosis. Endocytosisis often initiated by the binding of a ligand to a cell surface receptorand results in the uptake of extracellular materials, including fluid,dissolved solutes, and particulate matter. All eukaryotic cells undergoa continuous process of vesicle formation at the cytoplasmic side of theplasma membrane. Following uptake, vesicles are directed to any of anumber of cellular locations. The pathway and ultimate destination aredirected by a variety of signal motifs present in the cytoplasmic,transmembrane and extracellular domains of the proteins located on thevesicles, and, in some cases, by the non-protein membrane components ofthe vesicles.

[0200] Transcytosis refers to a process in which the vesicles aretransported from one side of a polarized cell (e.g., an epithelial cellor an endothelial cell) to the other side. The vesicle docks and fuseswith the plasma membrane and the contents are emptied to theextracellular compartment. Polarized cells in which such transportoccurs are present in many tissues. In all epithelial layers, the layersof cells separating the body from the outside world, the cells arepolarized. Epithelial cell layers are characterized by the presence oftight junctions that form an effective seal between all the cells of thelayer. It is this seal that divides the cells into an apical (outside)and a basal (inside) surface. The areas between the cells on the insideside are lateral; hence, the entire inside surface of the epithelialcell is known as the “baso-lateral” surface.

[0201] The reagents provided herein can be utilized to conduct screensof proteins to identify receptors that are involved in endocytosis andtranscytosis pathways. The preparation of the reagents for use in suchassays closely parallel the methods described supra. In general, a cDNAdisplay library is prepared as set forth above from cells involved inendocytosis (essentially any cell) or transcytosis (polarized cells suchas epithelial or endothelial cells). An antibody collection against allsurface proteins or apical surface proteins is prepared using suchproteins as immunogens. The cDNA library is subsequently incubated withthe antibody collection to form a population of replicable geneticpackage/antibody reagents; unbound antibodies are washed away.

[0202] These reagents can then be utilized to conduct a variety of invitro or in vivo assays to identify reagents bearing an antibody capableof binding to a cell surface receptor and causing transport of thereagent into or through a cell. By determining the sequence of theheterologous sequence of such reagents, one can determine the identityof the receptor involved in transport.

[0203] For example, certain in vitro assays involve growing polarizedcells (e.g., CaCo cells) as a monolayer on a semipermeable membrane thatcontains pores sufficiently large to allow the passage of reagentstherethrough and which divides an apical compartment from a basalcompartment. The replicable genetic package/antibody reagents arecontacted with the apical side of the cells in the apical compartment.The reagents are allowed to remain in contact for a period of timesufficient to allow transport through the cells. Reagents that have beentransported into the basal compartment are subsequently collected. Suchreagents display antibodies capable of interacting with a cell surfacereceptor that causes transcytosis. By determining the sequence of theheterologous sequence of such reagents, one can determine the identityof the receptor involved in the transport.

[0204] A number of other in vitro and in vivo methods for assaying forendocytosis and transcytosis that can be utilized with the reagentsprovided herein are described in PCT publication WO 01/23619.

[0205] The following examples are provided to illustrate certain aspectsof the methods, antibody/package reagents and arrays described hereinand are not to be construed so as to limit the scope of the invention.

EXAMPLE 1 Capture and Display of Polyclonal Antibodies Against the hTNFReceptor on T7 Phage Displaying hTNF Receptor cDNA Fragments

[0206] I. General

[0207] A cDNA fragment library of the human type I TNF receptor(hTNFR-1) extracellular domain was cloned into the mid-copy expressionT7 display vector T7Select10-3b (Novagen). This expression systemsproduces T7 phage particles displaying, on average approximately 5-15copies/phage of the protein encoded by the cloned cDNA sequence fused tothe T7 capsid protein (gene 10). A goat polyclonal antibody againstrhTNFR-1 was obtained from R&D Systems, as was a preparation of solublerhTNFR-1 extracellular domain protein.

[0208] An anti-rhTNFR-1 phage-displayed antibody reagent was created bymixing a T7-displayed rhTNFR-1 cDNA epitope fragment with the polyclonalantibody for use in a series of feasibility experiments demonstrating:(a) free anti-rhTNFR-1 binding sites are carried by the phage, (b) thebinding sites are available for binding to proteins external to thephage, (c) phage displaying the antibodies can be captured on animmobilized form of the target protein and detected with severalphage-specific and target specific reagents, (d) the extent to which therhTNFR-antibody displayed phage react with other proteins in a complexcellular preparation of proteins, and (e) the ability to detect thespecific target protein in a mixture of non-target proteins.

[0209] II. Preparation of the T7 hTNFR cDNA Fragment Library

[0210] A cDNA encoding the extracellular domain of the human type I TNFreceptor was cloned from human liver cDNA (obtained from Clontech) byPCR using gene specific primers. The TNFR cDNA was amplified and thendigested with DNaseI in the presence of MnCl₂ to produce randomdouble-stranded breaks in the DNA. Fragments of 100 to 300 base pairs inlength were gel purified and treated with Klenow fragment of DNApolymerase I to create blunt ends. The cDNA fragments were then ligatedto prepared vector arms of the T7Select10-3b display vector (Novagen) toobtain a library of inserts, approximately ⅙^(th) of which are in thecorrect orientation and same translational reading frame as the 10Bcapsid protein. The resulting DNA was incubated with a T7 in vitropackaging extract, and the phage products were amplified by infecting aculture of BLT5615 host cells. The culture was incubated with shaking at37° C. for 1-3 hours until lysis was observed, followed bycentrifugation at 8,000×g for 10 minutes to clarify the lysate ofresidual bacterial cells and debris.

[0211] T7 phage particles were further purified from the clearedsupernatant by precipitation with polyethylene glycol (PEG 8000) andbanding in a CsCl step gradient. Briefly, phage were precipitated fromthe supernatant by adding PEG 8000 to a final concentration of 10%(w/v), incubating on ice for 1 hour, followed by centrifugation at8,000×g for 15 minutes. Phage were extracted from the PEG pellet in 1MNaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, and the concentrated phagesolution was layered atop four steps of different density CsCl solutionsin a clear ultracentrifuge tube. The four CsCl layers were made bymixing a stock solution of 62.5% CsCl in water with TE buffer (10 mMTris-HCl, pH 8.0, 1 mM EDTA) in the following ratios: 1:2, 1:1, 2:1, 1:0CsCl:TE. Successively denser solutions were underlayed in the tube andthe concentrated phage solution was layered on top of the CsCl steps.The tubes were centrifuged at room temperature for 60 minutes at 35,000rpm in a Beckman SW41 rotor. Following centrifugation, the turbid bandof phage particles above the 2:1 layer was removed by piercing the sideof the tube with a syringe needle. Recovered phage particles were thendialyzed in PBS and stored at 4° C. or at −80° C. following the additionof 8% glycerol.

[0212] III. Screening T7 hTNFR cDNA Fragment Library Against the GoatPolyclonal anti-rhTNFR Antibody

[0213] To isolate T7 phage clones displaying hTNFR epitope fragmentsreactive with the goat polyclonal antibody against hTNFR (BAF225), thefragment library was first selected on immobilized antibody. Briefly,six wells of a 96-well microtiter plate were coated with 5 ugNeutrAvidin biotin binding protein and then blocked with PBS/1% BSA. 1ug of biotinylated polyclonal goat anti-hTNFR antibody was added to eachwell and incubated at 4° C. for 1 h, followed by washing with PBS. Analiquot of the T7 hTNFR cDNA fragment library was added to each antibodycoated well (approximately 10⁹ TU/well) and incubated at 4° C. for 1 h.The wells were washed extensively with PBS to remove unbound phage, andphage bound to the antibody were recovered by adding PBS/1% SDS to eachwell. The eluates were then combined and titered on a lawn of BLT5615cells.

[0214] Goat anti-hTNFR reactive phage clones were isolated from therecovered phage population by performing plaque lift analysis asfollows. Nitrocellulose membranes were placed on plaque-containingLB/Amp plates for approximately 1 min. The filters were removed andblocked for 1 h at room temperature with PBS/1% BSA, washed severaltimes with PBS, and incubated for 1 h at 4° C. with the biotinylatedgoat anti-hTNFR antibody diluted to 1 ug/ml in PBS/0.05% Tween 20/0.1%BSA (PBST/0.1% BSA). Filters were then washed with PBS and incubatedwith horseradish peroxidase-conjugated streptavidin (1 ug/ml inPBST/0.1% BSA) for 1 h at 4° C. Phage plaques stained with thebiotinylated antibody and HRP-streptavidin were detected using thecolorometric horseradish peroxidase substrate 3,3′5,5′-tetramethylbenzidine (TMB). Six positive plaques were picked andamplified by infecting cultures of BLT5615 cells; bacterial debris wasremoved from the lysates by centrifugation and the phage containingsupernatants were further characterized in a phage ELISA.

[0215] For each clone, duplicate wells of a 96-well plate were coatedwith NeutrAvidin and BAF225 as described above, as well as negativecontrol wells that contained only NeutrAvidin or were blocked withPBS/1% BSA. 50 ul of phage supernatant was added to each well andincubated for 1 h at 4° C. The wells were washed with PBS and boundphage were detected by adding 50 ul of rabbit anti-T7 phage antiseradiluted 1:5000 in PBS/0.1% BSA. The anti-phage antibody was detectedusing a horseradish peroxidase conjugated goat anti-rabbit IgG antibody,followed by addition of ABTS development buffer. The amount ofhorseradish peroxidase activity in each well was then measured byreading the absorbance at 405 nm with a microtiter plate reader. Phagedisplaying epitopes reactive with BAF225 were specifically captured onthe antibody coated wells (FIG. 2). Sequencing the TNFR cDNA fragmentsdisplayed by the positive clones revealed that multiple epitopes in theextracellular domain of TNFR are recognized by the polyclonal antibodies(FIG. 3).

[0216] IV. Capture of hTNFR Epitope T7 Phage on Immobilized rhTNFR witha Polyclonal Anti-TNFR Antibody

[0217] 1 ug of an anti-hTNFR monoclonal antibody (MAb 625) was added towells of a microtiter plate and incubated for 1 h at 37° C. The wellswere washed several times with PBS and blocked by adding 300 ul of PBScontaining 1% BSA. The plate was washed and 0.1 ng of soluble rhTNFR-1diluted in PBS/0.1% BSA was added to each well and incubated for 1 h at4° C. Following a wash with PBS either 100, 10, or 1 ng of thepolyclonal goat anti-hTNFR antibody (AF225) was added to the wells andincubated for 1 h at 4° C. The plate was again washed with PBS and 10⁹pfu of BAF225-3 phage which display an epitope that binds to the goatpolyclonal anti-hTNFR antibody were added to the wells and incubated for1 h at 4° C. Control wild type T710-3b phage that do not display ananti-TNFR epitope were added to other wells to serve as a negativecontrol. The plate was washed several times with PBS and bound phagewere detected by adding a polyclonal anti-T7 rabbit antisera followed byan anti-rabbit IgG HRP conjugate to each well. Bound antibody wasdetected by the addition of ABTS substrate solution and the absorbancewas measured using a microtiter plate reader.

[0218] The TNFR epitope phage clone BAF225-3 was specifically capturedby the polyclonal anti-hTNFR antibody only in the wells containingimmobilized hTNFR, thereby demonstrating the formation of a complexbetween epitope phage, antibody and target protein (FIG. 4).

EXAMPLE 2 Detection of Anti-hTNFR T7 Phage-Displayed Antibody on aWestern Blot of hTNRF

[0219] 1 ug of soluble human TNFR was loaded on a 4-12% NuPAGE gel(Invitrogen) and electrophoresed for 45 min at 200V, followed bytransfer of the protein to a nitrocellulose membrane. The filter wasblocked by incubating for 1 h at 4° C. in TBS/5% milk/1% goat serum/0.1%Tween 20. The anti-hTNFR antibody-phage complex was formed by combining10¹⁰ pfu of purified BAF225-3 phage with 10 ug of the goat anti-hTNFRpolyclonal antibody (AF225) and incubating for 1 h at 4° C. Thepreformed antibody-phage complex was then diluted in 10 ml of TBS/1%milk/0.2% goat serum/0.1% Tween 20 and added to the TNFR blot. Followingan overnight incubation at 4° C., the blot was washed several times withPBS/0.1% Tween 20 and then incubated with a polyclonal anti-T7 rabbitantisera followed by an anti-rabbit IgG HRP conjugate. Bound antibodywas detected by incubating the blot in TMB substrate solution, whichproduces a colored precipitate on the blot where enzyme activity islocated (see FIG. 5A).

[0220] The approximately 21-kD soluble TNFR protein band was clearlydetected with the anti-T7 phage antibody, indicating that the anti-TNFRphage displayed antibody complex was bound to the target protein (FIG.5B). The large amount of BSA present in the preparation of soluble TNFR,which served as a negative control for non-specific binding, showed nostaining.

EXAMPLE 3 Capture and Recovery of Anti-hTNFR T7 Phage-Displayed Antibodyon Western Blot of hTNFR

[0221] A western blot of soluble hTNFR was prepared as described in theExample 2 to determine if infective phage particles bound to the targetprotein could be recovered from the blot (see FIG. 6A). To prepare theanti-hTNFR phage antibody complex, a 1:10 mixture of epitope phage(BAF225-3) and wild type T7 10-3b phage was incubated with 10 ug of thegoat anti-hTNFR polyclonal antibody (AF225) for 1 h at 4° C. Thepreformed antibody-phage complex was then diluted in 10 ml of TBS/1%milk/0.2% goat serum/0.1% Tween 20 and added to the TNFR blot. Followingan overnight incubation at 4° C. the blot was washed extensively withPBS/0.1% Tween 20. Two regions of the blot, one corresponding to thelocation of the soluble TNFR protein band and another that served as anegative control, were excised and placed into PBS containing 1% SDS toelute the bound phage. To determine the number of phage recovered fromeach sample, the eluates were titered by infecting BLT 5615 cells andplating on LB/AMP plates.

[0222] Approximately 100-fold more phage were recovered from the TNFRband as from the negative control (FIG. 6B). To determine whatproportion of these phage displayed the target epitope, a plaque liftwith the anti-epitope antibody (AF225) was performed. 95% of the phagefrom the TNFR band were target phage, representing an enrichment of190-fold relative to negative phage, while in the negative control bandapproximately 10% were target phage, identical to the fraction in theinput population. The results indicate that specific phage-antibodycomplexes form and persist, and retain free epitope-binding sitescapable of forming stable interactions with the cognate target protein.

EXAMPLE 4 Detection of hTNFR in a Complex Mixture of Proteins Using T7Phage-Displayed Anti-hTNFR Antibody

[0223] 50 ng of soluble hTNFR was combined with 2 ug of MDCK cell totalprotein extract and electrophoresed on a 4-12% NuPAGE gel as describedin Example 2. The separated proteins were transferred to anitrocellulose filter and the blot was blocked overnight at 4° C. inTBS/5% milk/1% goat serum/0.1% Tween 20. The anti-hTNFR antibody-phagecomplex was formed by combining 10¹⁰ pfu of purified BAF225-3 phage with10 ug of the goat anti-hTNFR polyclonal antibody (AF225) and incubatingfor 1 h at 4° C. The preformed antibody-phage complex was then dilutedin 10 ml of TBS/1% milk/0.2% goat serum/0.1% Tween 20 and added to theprotein blot. Following an overnight incubation at 4° C., the blot waswashed several times with PBS/0.1% Tween 20 and then incubated with apolyclonal anti-T7 rabbit antisera followed by an anti-rabbit IgG HRPconjugate. The blot was then developed in TMB substrate solution todetermine the location of immunoreactive protein bands (see FIG. 7A).

[0224] The approximately 21-kD soluble TNFR protein band was clearlydetected with the anti-T7 phage antibody, indicating that the anti-TNFRphage displayed antibody complex was bound to the target protein (FIG.7B). No bands were detected on control replica blots probed withoutepitope phage or the polyclonal anti-TNFR antibody.

EXAMPLE 5 Capture and Display of Antibodies Against the hTNF Receptor onfd Phage Displaying hTNF Receptor cDNA Fragments

[0225] I. General

[0226] A cDNA fragment library of the human type I TNF receptor(hTNFR-1) extracellular domain was cloned into a pVIII phagemidexpression vector. Depending on the induction conditions used duringphage growth, this system produces filamentous phage particlesdisplaying approximately 10 to several hundred copies of the proteinencoded by the cloned cDNA sequence fused to the major coat proteinpVIII. An anti-rhTNFR-1 phage-displayed antibody reagent was created bymixing a fd-displayed rhTNFR-1 cDNA epitope fragment with a polyclonalantibody for use in a series of feasibility experiments demonstrating:(a) free anti-rhTNFR-1 binding sites are carried by the phage, (b) thebinding sites are available for binding to proteins external to thephage, (c) phage displaying the antibodies can be captured on animmobilized form of target protein and detected with severalphage-specific and target specific reagents.

[0227] II. Preparation of the fd hTNFR cDNA Fragment Library

[0228] A cDNA encoding the extracellular domain of the human type I TNFreceptor was cloned from human liver cDNA by PCR using gene specificprimers. The amplified cDNA was digested with DNaseI in the presence ofMnCl₂ to produce random double-stranded breaks in the DNA. Fragments of100 to 300 base pairs in length were gel purified and treated withKlenow fragment of DNA polymerase I to create blunt ends. The cDNAfragments were then ligated to the pVIII phagemid vector (p8cDNA) toobtain a library of inserts of which some are in the correct orientationand same translational reading frame as the major (pVIII) coat protein.The resulting DNA was electroporated into E. coli MC1061 F′ cells, andthe cells were grown without selection at 37° C. for 1 h. The cells werethen added to a larger culture of medium containing ampicillin to selectfor the presence of phagemid, and glucose to repress the expression ofthe fusion protein. The cells were grown for three to four hours andwere then infected with the helper phage M13KO7. Kanamycin was added tothe culture to select for the presence of the helper phage and arabinosewas added to induce the expression of the recombinant fusion protein.Following overnight incubation at 37° C., the culture was centrifuged at12,000×g for 15 minutes to pellet the bacterial cells, and the phagecontaining supernatant was transferred to a new bottle. Fd phageparticles were purified from the supernatant by precipitation withpolyethylene glycol (PEG 8000) followed by centrifugation at 12,000×gfor 15 minutes. The supernatant was removed and the phage pellet wasresuspended in PBS.

[0229] III. Screening fd hTNFR cDNA Fragment Library Against the GoatPolyclonal anti-rhTNFR Antibody

[0230] To isolate fd phage clones displaying hTNFR epitope fragmentsreactive with the goat polyclonal antibody against hTNFR (BAF225), thefragment library was first selected on the immobilized antibody. Sixwells of a 96-well microtiter plate were coated with 5 ug NeutrAvidinbiotin binding protein and then blocked with PBS/1% BSA. 1 ug ofbiotinylated polyclonal goat anti-human hTNFR antibody was added to eachwell and incubated at 4° C. for 1 h, followed by washing with PBS. Analiquot of the fd hTNFR cDNA fragment library was added to each antibodycoated well (approximately 10⁹ TU/well) and incubated at 4° C. for 1 h.The wells were washed extensively with PBS to remove unbound phage, andphage bound to the antibody were recovered by adding an acid elutionbuffer (0.1 M HCl, pH 2.2 with glycine, 0.1% BSA), followed byneutralization with an equal volume of 2M Tris base (pH unadjusted). Theeluates were combined and titered on K91 recA cells.

[0231] Random clones from the eluate were grown and tested individuallyin a phage ELISA against the immobilized polyclonal antibody. For eachclone duplicate wells of a 96-well plate were coated with NeutrAvidinand BAF225 as described above, as well as negative control wells thatcontained only NeutrAvidin or were blocked with PBS/1% BSA. 50 ul ofphage supernatant was added to each well and incubated for 1 h at 4° C.The wells were washed with PBS and bound phage were detected by adding50 ul of an anti-fd antibody conjugated to HRP diluted 1:5000 inPBS/0.1% BSA. The anti-phage antibody was detected by addition of ABTSdevelopment buffer. The amount of horseradish peroxidase activity ineach well was then measured by reading the absorbance at 405 nm with amicrotiter plate reader. Phage displaying epitopes reactive with BAF225were specifically captured on the antibody coated wells (FIG. 8).Sequencing the TNFR cDNA fragments displayed by the positive clonesrevealed that multiple epitopes in the extracellular domain of TNFR arerecognized by the polyclonal antibodies (FIG. 9).

[0232] IV. Capture of hTNFR Epitope fd Phage on Immobilized rhTNFR witha Polyclonal anti-TNFR Antibody

[0233] 1 ug of an anti-hTNFR monoclonal antibody (MAb 625) was added towells of a microtiter plate and incubated for 1 h at 37° C. The wellswere washed several times with PBS and blocked by adding 300 ul of PBScontaining 1% BSA. The plate was washed and 0.1 ng of soluble rhTNFR-1diluted in PBS/0.1% BSA was added to each well and incubated for 1 h at4C. Following additional washes with PBS, either 100, 10, or 1 ng of apolyclonal goat anti-hTNFR antibody (AF225) was added to the wells andincubated for 1 h at 4° C. The plate was again washed with PBS and 10⁹TU of BAF225-10 phage which display an epitope that binds to the goatpolyclonal anti-hTNFR antibody were added to the wells and incubated for1 h at 4° C. Control wild type p8 phage that do not display an anti-TNFRepitope were added to other wells to serve as a negative control. Theplate was washed several times with PBS and bound phage were detected byadding an anti-fd antibody conjugated to HRP that was diluted 1:5000 inPBS/0.1% BSA. Bound antibody was detected by the addition of ABTSsubstrate solution and the absorbance was measured using a microtiterplate reader.

[0234] The TNFR epitope fd phage clone BAF225-10 was specificallycaptured by the polyclonal anti-hTNFR antibody only in the wellscontaining immobilized hTNFR, thereby demonstrating the formation of acomplex between epitope phage, antibody and target protein (FIGS. 10Aand 10B).

EXAMPLE 6 Detection of anti-hTNFR fd Phage-Displayed Antibody on WesternBlot of hTNRF

[0235] 1 ug of soluble human TNFR was loaded on a 4-12% NuPAGE gel(Invitrogen) and electrophoresed for 45 min at 200V, followed bytransfer of the protein to nitrocellulose. The filter was blocked byincubating for 1 h at 4° C. in PBS containing 1% BSA. The anti-hTNFRantibody-phage complex was formed by combining 10¹⁰ TU of purifiedBAF225-10 phage with 10 ug of the goat anti-hTNFR polyclonal antibody(AF225) and incubating for 1 h at 4° C. The preformed antibody-phagecomplex was then diluted in 10 ml of PBS/0.1% BSA and added to the TNFRblot. Following an overnight incubation at 4° C., the blot was washedseveral times with PBS and then incubated with an anti-fd antibody HRPconjugate. Bound antibody was detected by incubating the blot in TMBsubstrate solution, which produces a colored precipitate on the blotwhere enzyme activity is located (see FIG. 11A).

[0236] The approximately 21-kD soluble TNFR protein band was clearlydetected with the anti-fd phage antibody, indicating that the anti-TNFRphage displayed antibody complex was bound to the target protein (FIG.11B). No bands were detected on a second blot that was incubated withepitope phage minus the polyclonal anti-hTNFR antibody. The large amountof BSA present in the preparation of soluble TNFR, which served as anegative control for non-specific binding, also showed no staining.

EXAMPLE 7 Identification of Proteins Separated by 2-D electrophoresis

[0237] I. Overview

[0238] A polyclonal antibody population is produced against the proteincomplement of MDCK cells by immunization of rabbits with a mixture ofproteins prepared from 5 day differentiated MDCK cell cultures. cDNA isprepared from 2, 3, and 4 day differentiated MDCK cells and cloned intothe T7 low and mid-copy expression vectors and fd pIII and pVIIIphagemid expression vectors to produce a cDNA display library of theproteins expressed by MDCK cells. The library and the polyclonal Igpreparation are mixed to produce the captured anti-MDCK antibody displaylibrary to be used in the following set of experiments.

[0239] II. Experimental

[0240] A. Isolation of Protein from MDCK Cells

[0241] 1) Growth of MDCK Cells:

[0242] Low passage number MDCK cells are grown in DMEM supplemented with10% FBS and antibiotics (Kanamycin 100 ug/ml; Penicillin 0.5 units/ml;Streptomycin 0.5 ug/ml) to approximately 80% confluence.

[0243] Cells are removed from the dishes with trypsin/EDTA.

[0244] Cells are seeded at confluent density (approximately 5×10⁵cells/cm²) onto tissue culture plates or 0.4 um permeable supports(transwells: 24 mm and 75 mm diameter).

[0245] Cells are returned to the incubator for 5 days, changing mediumevery other day, to establish differentiated monolayers

[0246] 2) Preparation of Whole Cells and Protein Fractions for Use asImmunogens

[0247] As described supra, proteins used as immunogens can be of avariety of different types (including, but not limited to, proteinfractions isolated from cells or tissues, proteins expressed from a cDNAdisplay library, and random populations of peptides). These proteins canbe used as immunogens to prepare antibodies using a number of differentformats as described above. Furthermore, a number of approaches can beused to generate a distinct population of proteins from whole cells touse as immunogens for the preparation antibodies. This means focusedantibody populations can be prepared from a number of different sourcesincluding, but not limited to, whole cells, cell surface proteins,proteins associated with various membrane fractions (e.g., proteins fromthe endoplasmic reticulum, Golgi, endosome, plasma membrane, and solubleproteins).

[0248] a) Isolation of Total Protein from Cells Grown on Plastic orPermeable Support:

[0249] Cells are grown on either a plastic or a permeable support (e.g.,Costar Transwell Filters) for an appropriate period of time (e.g., 5days past confluence for differentiated MDCK cells, 14-21 days forpolarized Caco-2 cells, or to confluence for non-polarized cells).Filters/plates are placed on ice and washed 3× with ice-cold Ringer'ssaline (10 mM HEPES, pH 7.4, 150 mM NaCl, 7.2 mM KCl, 1.8 mM CaCl₂). Thecells are then extracted by adding extraction buffer [0.5% Triton X-100,300 mM Sucrose, 10 mM PIPES, pH 6.8, 50 mM NaCl, 3 mM MgCl₂ and1×protease inhibitors (antipain (10 μg/ml); leupeptin (10 μg/ml); andpepstatin A (10 μg/ml) plus 1 mM pefablock)] to both apical and basalchambers and rocking gently for 30 min at 4° C. The lysates are thentransferred to a clean tube (combining multiple samples if necessary).The filters/plates are scraped and the material collected is combinedwith the lysates. The samples are vortexed and placed on ice for 10 min.The samples are then transferred to a microfuge tube and centrifuged at20,000×g for 10 min. The supernatant is then transferred to a new tube.The protein concentration of the sample is measured and adjusted to 1mg/ml. Note: alternative detergents can be used for extraction (forexample 1% SDS; 2% CHAPS; 0.5% TX-100 and 1% Dexoycholate and 0.1% SDS),but must be dialyzed against an isotonic salt solution with minimaldetergent present to maintain solubility of integral membrane proteins.

[0250] b) Isolation of Domain Specific Plasma Membrane Protein Fractionsfrom Cells Grown on Permeable Supports:

[0251] Cells are grown on a permeable support (e.g., Costar TranswellFilters) for an appropriate period of time (e.g., 5 days past confluencefor differentiated MDCK cells, 14-21 days for polarized Caco-2 cells).Filters are placed on ice and washed 3× with ice-cold Ringer's saline(10 mM HEPES, pH 7.4, 150 mM NaCl, 7.2 mM KCl, and 1.8 mM CaCl₂). Cellsare then treated with a membrane impermeant biotinylation reagent (forexample EZ-Link sulfo-NHS-S-S-Biotin, EZ-Link sulfo-NHS-LC-LC-Biotin).Biotinylation reagent (made up as 400×stock in DMSO) diluted to 750 uMin Ringer's saline (10 mM HEPES, pH 7.4, 150 mM NaCl, 7.2 mM KCl, 1.8 mMCaCl₂) is then applied. Apply 1 ml/4 ml biotin solution apical or 2 ml/6ml basal for 24 mm/75 mm transwells, respectively, and rocked at 4° C.for 20 min. Ringer's saline is applied to the opposite chamber (1 ml/4ml apical or 2 ml/6 ml basal for 24 mm/75 mm transwells, respectively)if doing domain specific cell surface labeling. After 20 min, aspirateand then repeat the treatment with fresh biotin solution, as above.Cells are extracted in 1 ml (24 mm)/10 ml (75 mm) extraction buffer[0.5% Triton X-100, 300 mM Sucrose, 10 mM PIPES, pH 6.8, 50 mM NaCl, 3mM MgCl₂ and 1×protease inhibitors (antipain (10 μg/ml), leupeptin (10μg/ml) plus 1 mM pefablock)] to both apical and basal-lateral sides androcked gently for 30 min at 4° C. An equal volume of extraction bufferis applied to both apical and basal-lateral sides and rocked gently for30 min at 4° C. The lysates are then transferred to a clean tube(combine multiple samples if necessary). The filters are scraped and thematerial collected is combined with the lysates. The samples arevortexed and placed on ice for 10 min. Lysates are transferred into 1.5ml tubes (1 ml/tube) and 50 ul Pansorbin added to each and mixed 30 minat 4° C. to pre-clear (pansorbin pre-washed 2× and resuspended inextraction buffer and 1×P.I. and 1 mM pefablock). The samples are thencentrifuged at 20,000×g for 10 min. The supernatant is then transferredto a new tube. 100 ul of neutravidin agarose is added to each tube andincubated overnight at 4° C. on rotator (neutravidin agarose pre-washed2× and resuspended in extraction buffer and 1×P.I. and 1 mM pefablock).Samples are centrifuged at 2000×g for 1 min. Beads are washed 2× withHigh Stringency buffer (0.1% SDS, 1% deoxycholate, 0.5% Triton X-100, 20mM Tris pH 7.5, 120 mM NaCl, 25 mM KCl, 5 mM EDTA and 5 mM EGTA), 2×with High Salt buffer (0.1% SDS, 1% deoxycholate, 0.5% Triton X-100, 20mM Tris pH 7.5, 120 mM NaCl, 25 mM KCl, 5 mM EDTA, 5 mM EGTA and 1 MNaCl), and 2× with Low Salt buffer (2 mM EDTA, 10 mM Tris pH 7.5). Beadsare then resuspended in an isotonic buffer and proteins left on beadsare used directly as immunogens in the case of non-reduciblebiotinylation reagents. If necessary, when using a reduciblebiotinylation reagent (for example EZ-Link sulfo-NHS-S-S-Biotin),proteins can be eluted from beads by addition of 200 ul/tube ofHomogenization Buffer (20 mM HEPES/KOH, pH 7.2, 90 mM KOAc, 2 mMMg(OAC)₂, 250 mM Sucrose), 5% BME, 1×protease inhibitors, and 1 mMpefablock for 1 h at room temperature.

[0252] c) Preparation of Whole Cells (Live and Fixed) for Use asImmunogens:

[0253] The protocol used for MDCK cells but can be adapted to other celltypes as well. Cells were harvested and plated at sufficient density toreach approximately 70% confluence 2 days after plating (sufficient timefor the full compliment of cell surface proteins to be expressedproperly, but brief enough to allow removal of cells from flask). Aftertwo days, cells were washed 3× with PBS and released from the flaskswith versene to ensure that cell surface proteins were not compromised.The cells were then pelleted by low speed centrifugation and either: 1)resuspended in freezing medium (50% DMEM, 40% FBS and 10% DMSO) andslowly frozen and stored in liquid nitrogen to maintain cell viabilityor 2) washed 4× with PBS, fixed in 4% paraformaldehyde for 30 min onice, then washed 4× with TBS (10 mM Tris pH 7.4, 120 mM NaCl) plus 50 mMNH₄Cl. In both cases, cells were resuspended 1×10⁷ cells/vial for eachinoculation of rabbits or 1×10⁶ cells/vial for each inoculation of mice.FIGS. 12A and 12B show lysates from MDCK, NRK 49F, NRK 52E, and Caco-2cells separated by SDS-PAGE and probed with various bleeds from 1 ofthree rabbits inoculated with “live MDCK cell prep” (FIG. 12A) or bleedsfrom 1 of three rabbits inoculated with “fixed MDCK cell prep” (FIG.12B).

[0254] d) Preparation of Plasma Membrane Enriched Fraction for Use asImmunogens:

[0255] The protocol used for differentiated MDCK (5 days pastconfluence) and Caco-2 cells (grown for 19-21 days past confluence)grown on 75 mM Transwells can be adapted to other cell types grown onplastic or permeable supports. Cells were placed on ice and washed 3×with ice-cold Ringer's saline (10 mM HEPES, pH 7.4, 150 mM NaCl, 7.2 mMKCl, 1.8 mM CaCl₂) and 1× with homogenization buffer (20 mM HEPES/KOH pH7.2, 90 mM KOAc, 2 mM Mg(OAc)₂, 250 mM sucrose). Cells were scraped fromthe filter in homogenization buffer containing protease inhibitors(antipain (10 μg/ml), leupeptin (10 μg/ml) plus 1 mM pefablock). Cellswere combined in a 50 ml conical tube and centrifuged at 2000×g for 10min at 4° C. Cells were then resuspended in a minimal volume ofhomogenization buffer containing protease inhibitors. The cells weregently fragmented by passing the sample through a ball bearinghomogenizer multiple times (minimal clearance between the bore of thechamber and the ball bearing ensure that the cell are fragmented withoutdisrupting organelles). The homogenate was transferred to microfugetubes and centrifuged at 2000×g for 10 min. The supernatant wastransferred to a new microfuge tube. The pellet was resuspended in aminimal volume and re-homogenized as above. The second homogenate wastransferred to microfuge tubes and centrifuged at 2000×g for 10 min topellet any remaining unbroken cells and nuclei. The supernatant from thesecond homogenate was combined with the supernatant from the firsthomogenization. The sample was then centrifuged at 10,000×g for 10 minto remove large membrane components (for example mitochondria andendoplasmic reticulum). The supernatant was then centrifuged at100,000×g for 45 min to produce an enriched plasma membrane fraction.The supernatant was discarded and the pellet resuspended inhomogenization buffer containing protease inhibitors. A protein assaywas preformed to determine the protein concentration in each sample.Based on the results from the protein assay, the samples were examinedby SDS-PAGE (FIGS. 13A and 13B). The results from the SDS-PAGE analysiswere used to estimate the actual protein concentration in each sampleprior to its use as an inoculum. FIGS. 14A and 14B show lysates fromMDCK, NRK 49F, NRK 52E, and Caco-2 cells separated by SDS-PAGE andprobed with various bleeds from 1 of three rabbits inoculated with “MDCK100k mem prep” (FIG. 14A) or bleeds from 1 of three rabbits inoculatedwith “Caco-2 100K membrane prep” (FIG. 14B).

[0256] B. Preparation of Rabbit Antibody Against Total MDCK ProteinPreparation

[0257] 2-3 rabbits are used for each antigen

[0258] pre-immune bleeds are collected from each rabbit and screenedagainst the antigen preparation

[0259] rabbit polyclonal antibodies are raised against proteinpreparations isolated as outlined above using a standard 94 day protocol(Covance):

[0260] 500 ug of antigen is used for the initial immunization and 250 ugof antigen for each subsequent boost

[0261] each rabbit is given four boosts based on a three-week cycle ofboost, in which a 50 ml sample of blood is collected 10 days after eachboost

[0262] following the fourth boost rabbits are exsanguinated

[0263] serum is collected from each bleed and titered against theantigen

[0264] serum from each production sample is then screened by westernblot against the antigen.

[0265] immunoreactive serum samples are affinity purified by isolationon protein-A Sepharose and salt elution

[0266] C. Preparation of cDNA from MDCK Cells

[0267] 1) Growth of cells: as above.

[0268] 2) Isolation of mRNA: MDCK cells are removed from the Transwellfilter by scraping and the cell suspension is transferred to acentrifuge tube. The cells are pelleted by centrifugation at 300×g for10 minutes. Total mRNA is isolated by disrupting the cells in thepresence of guanidine isothiocyanate followed by the addition of ethanolto provide the appropriate conditions for binding RNA to thesilica-based membrane in the spin column (according to manufacturersprotocols for RNeasy kit obtained from Qiagen). The column is washedseveral times and purified RNA is eluted with water. mRNA is isolated bymixing the total RNA preparation with an equal volume of 20 mM Tris-Cl,pH 7.5, 1M NaCl, 2 mM EDTA, 0.2% SDS, followed by incubating the samplefor 3 min at 70° C. The sample is then incubated at room temperaturewith dC₁₀T₃₀ oligonucleotides that are covalently attached to thesurface of polystyrene-latex particles (Oligotex columns obtained fromQiagen). The column is washed several times and poly A+ mRNA is elutedin 5 mM Tris-HCl, pH 7.5.

[0269] 3) Synthesis of cDNA: Poly A+ RNA is primed with oligo (dT) orHindIII random hexamer primers d(N₆TT) in the appropriate buffer andfirst strand synthesis is initiated by the addition of dNTPs and MMLVreverse transcriptase. RNAse H and DNA polymerase I are used for secondstrand synthesis, followed by treatment with T4 polymerase to blunt theends of the cDNA. First and second strand synthesis reactions arecarried out in the presence of 5-methyl dCTP, which protects anyinternal EcoRI and HindIII restriction sites from digestion insubsequent steps. EcoRI/HindIII directional linkers d(GCTTGAATTCAAGC)are ligated to the cDNA. The ligase is heat inactivated by incubating at70° C. for 10 minutes and the sample is cooled slowly to roomtemperature. The cDNA is then digested with EcoRI and HindIII and passedthrough a small gel filtration column to remove excess linkers and smallcDNA products (<300 bp).

[0270] 4) Preparation of cDNA fragments: Poly A+ RNA is primed witholigo (dT) or random hexamer primers d(N₆) in the appropriate buffer andfirst strand synthesis is initiated by the addition of dNTPs and MMLVreverse transcriptase. RNaseH and DNA polymerase I are used for secondstrand synthesis. The cDNA is then digested with DNaseI in the presenceof MnCl₂ to produce random double-stranded breaks in the DNA. Fragmentsof 100-300 base pairs in length are gel purified and treated with Klenowfragment of DNA polymerase I to create blunt ends.

[0271] D. Preparation of cDNA Library in T7

[0272] 1) Preparation of T7 vector DNA: CsCl purified T7 phage(described above) are dialyzed in 0.1 M NaCl, 0.1 M Tris-HCl, pH 8.0.The phage solution is then extracted three times with an equal volume ofphenol equilibrated with 0.1 M Tris (pH 7.5), followed by twoextractions with an equal volume of chloroform:isoamyl alcohol (24:1v/v). The final aqueous phase is dialyzed in TE buffer overnight at 4°C. 100 ug of vector DNA is digested with EcoRI and HindIII in theappropriate buffer overnight at 37° C. to clone the full-length cDNAs orEcoRV to clone the blunt ended cDNA fragments. The restriction enzymesare heat inactivated by incubating the sample at 65° C. for 20 minutes.The vector arms are dephosphorylated by the adding 50 units of calfintestine alkaline phosphatase to the DNA in the appropriate buffer andincubating at 37° C. for 1 hour. The sample is then extracted one timewith an equal volume of phenol:chloroform (1:1) and passed through asmall gel filtration column.

[0273] Ligation and purification: 2 ug of prepared vector arms are mixedwith a 2-fold molar excess of cDNA and ligated with T4 DNA ligaseovernight at 16° C. The ligation mixture is incubated with an in vitropackaging extract, and the phage products are used to infect a suitablebacterial host. Large amounts of phage can be prepared by infecting aculture of bacterial cells grown in M9TB to an OD₆₀₀ of 0.6-0.8 with thepackaging reaction. The culture is incubated with shaking at 37° C. for1-3 hours until lysis is observed. The lysate is clarified bycentrifugation at 8,000×g for 10 minutes. T7 phage are purified from thecleared supernatant by precipitation with polyethylene glycol (PEG 8000)followed by banding in a CsCl step gradient.

[0274] E. Preparation of cDNA Library in fd pIII and pVIII PhagemidExpression Vectors

[0275] Double-stranded Phagemid Vector DNA is isolated using QiagenPlasmid Maxi columns. 100 ug of each vector is digested with EcoRI andHind III in the appropriate buffer overnight at 37° C. The restrictionenzymes are heat inactivated by incubating the sample at 65° C. for 20minutes. To clone cDNA fragments the digested vectors are treated withKlenow fragment in the presence of all four dNTPs to create blunt ends.The vectors are dephosphorylated by adding 50 units of calf intestinealkaline phosphatase to the DNA in the appropriate buffer and incubatingat 37° C. for 1 hour. The samples are extracted one time with an equalvolume of phenol:chloroform (1:1) and passed through a small gelfiltration column.

[0276] 10 ug of each vector is mixed with a 2-fold molar excess of cDNAand ligated with T4 DNA ligase overnight at 16° C. Each ligation mix iselectroporated into E. coli MC1061 F′ cells, and the cells are grownwithout selection at 37° C. for 1 hour. The cells are then added tolarger cultures of medium containing ampicillin to select for thepresence of phagemid, and glucose to repress the expression of thefusion protein. The cells are grown for three to four hours(approximately 10-doublings) and are then infected with helper phage.Kanamycin is added to cultures to select for the presence of helperphage and arabinose is added to induce the expression of the recombinantfusion protein. Following an overnight incubation at 37° C., thecultures are centrifuged at 12,000×g for 15 minutes to pellet thebacterial cells, and the phage containing supernatants are transferredto new bottles. Fd phage are purified from each supernatant byprecipitation with polyethylene glycol (PEG 8000) followed bycentrifugation at 12,000×g for 15 minutes. The supernatant is removedand the phage pellet is resuspended in PBS.

[0277] F. Preparation of cDNA Expression Library Fused to GST forImmunization

[0278] 1) cDNA prepared as described above is digested with the EcoRIand HindIII and inserted into an expression vector that places the cDNAin the same translational reading frame as the gene forglutathione-S-transferase (GST) (pET vectors obtained from Novagen). Theplasmid construct is transformed into E. coli BL21 (DE3) to allowcontrolled expression of the GST fusion protein.

[0279] 2) Bacterial cells harboring the GST fusion vector are typicallygrown at 37° C. to an OD600 of ˜0.8 and IPTG is added to a finalconcentration of 1 mM to induce expression of the fusion protein. Thecells are grown for an additional 2.5 hours at 37° C. and pelleted bycentrifugation at 3000×x g for 10 minutes. Proteins are extracted fromthe cells using B-Per Reagent (Pierce) according to the manufacturer'sdirections. GST-cDNA fusion protein is purified from the total proteinpreparation by incubating the extract with immobilized glutathione.Bound fusion protein is released by the addition of elution buffercontaining reduced glutathione. The purified protein is dialyzed in PBSovernight at 4° C.

[0280] G. Use of GST-fused cDNA for Immunizing Rabbits

[0281] 1) Preparation of rabbit antibody against total GST-cDNAexpression library protein mixture

[0282] 2-3 rabbits are used for each antigen preparation

[0283] pre-immune bleeds are collected from each rabbit and screenedagainst the antigen preparation

[0284] rabbit polyclonal antibodies are raised against proteinpreparations isolated as outlined above using a standard 94 day protocol(Covance):

[0285] 500 ug of antigen is used for the initial immunization and 250 ugof antigen for each subsequent boost

[0286] each rabbit is given four boosts based on a three-week cycle ofboost, in which a 50 ml sample of blood is collected 10 days after eachboost

[0287] following the fourth boost rabbits are exsanguinated

[0288] serum is collected from each bleed and titered against theantigen

[0289] serum from each production sample is then screened by westernblot against the antigen

[0290] immunoreactive serum samples are affinity purified by isolationon protein-A Sepharose and salt elution

[0291] H. Capture of Antibodies on cDNA Library products Displayed onPhage

[0292] Phage particles from the MDCK cDNA display library are grown andpurified as described to produce approximately 10¹³ phage particles (orabout 10 ⁷ library equivalents from a library of 10⁶ independentrecombinants). Ig fraction from the anti-MDCK polyclonal is concentratedto less than or equal to 1 mg/ml protein, and 1-10 ml is mixed with 10¹³pfu of the library, incubated overnight at 4° C. Immediately before use,phage particles and free antibody are separated by pelleting the phageat 300,000×g for 30 min and resuspending in 10 ml (or less) PBS.

[0293] I. Separation and Blotting of Total Protein from MDCK Cells onSDS-PAGE and 2-D IEF/SDS-PAGE

[0294] 1. 1-D SDS-PAGE

[0295] pour 7.5% SDS-PAGE gels

[0296] add equal volume of SDS-PAGE sample buffer containing 200 mM DTTand 2% β-mercaptoethanol to each sample and mix

[0297] heat samples at 100° C. for 10 min

[0298] load samples on gel

[0299] run gels at 8 mA per gel overnight

[0300] Electrophoretic Protein Transfer to Immobilon PVDF

[0301] pre-wet membranes in 100% methanol

[0302] transfer proteins for 4 hr at 250 mA (->1 Amp-hr)

[0303] block blots in Tris buffered saline (TBS) (10 mM Tris-HCL, pH7.4, 120 mM)+5 % milk+1% goat serum+0.1% NaN₃ overnight at 4° C.

[0304] rinse blots once, then wash 1×5 min in TBS+0.1% Tween-20 wash 1×5min in HBS

[0305] Staining Blots with a Library of Phage-cDNA/Ab Complexes

[0306] probe blots with phage-displayed cDNA library/antibody reagent at10¹⁰-10¹² phage particles in TBS+1% milk+0.2% goat serum for 1 hour atroom temp on belly dancer

[0307] rinse blots 2×, then wash 4×10 min with TBS+0.1% Tween-20

[0308] wash 1×5 min with TBS

[0309] incubate blots with biotinylated anti-phage antibody (1 ug/ml)diluted in TBS+1% milk+0.2% goat serum for 1 hour at room temp on bellydancer

[0310] rinse blots 2×, then wash 4×10 min with TBS+0.1% % Tween-20

[0311] wash 2×5 min with TBS

[0312] incubate blots with fluorescein conjugated neutavidin diluted to1 ug/ml in TBS+1% milk+0.2% goat serum for 1 hour at room temp on bellydancer

[0313] rinse blots 2×, then wash 4×10 min with TBS+0.1% % Tween-20

[0314] wash 2×5 min with TBS

[0315] expose to Typhoon fluorescence array detector (MolecularDynamics, Sunnyvale, Calif.) as required

[0316] 2. 2-D Gels

[0317] Pour Isoelectric-focusing Gel

[0318] prepare acrylamide gel solution in side-arm flask. For 40 ml:

[0319] 21.86 g urea (9.1 M final)

[0320] 8 ml 10% (v/v) Triton X-100 (2% v/v final)

[0321] 3.6 ml water

[0322] 10 ml acrylamide/bis stock solution (30:0.8%)

[0323] 1.6 ml ampholytes pH 5-7 (Pharmacia, ampholynes)

[0324] (40% stock->1.6% final)

[0325] 0.4 ml ampholytes pH 3-10 (Pharmacia, pharmolytes)

[0326] (36% stock ->0.36% final)

[0327] de-gas solution

[0328] add 80 μl of 10% APS and 40 μl TEMED and mix

[0329] pour tube gels

[0330] Run Isoelectric-focusing Gel

[0331] prepare sample

[0332] thaw lysate

[0333] add dry urea to 9 M final concentration

[0334] volume increased 1.6-fold (to 373 μl)

[0335] want composition of sample following addition of lysis buffer tobe:

[0336] 9.1 M urea

[0337] 2% Triton X-100

[0338] 5% β-mercaptoethanol

[0339] 1.6% ampholytes pH 5-7

[0340] 0.36% ampholytes pH 3-10

[0341] add equal volume of “2X” lysis buffer containing:

[0342] 2.73 g urea (9.1 M in “2×”)

[0343] 2 ml 10% Triton X-100

[0344] 0.5 ml β-mercaptoethanol (10% in “2×”)

[0345] 0.4 ml ampholytes pH 5-7 (3.2% in “2×”)

[0346] 0.1 ml ampholytes pH 3-10 (0.72% in “2×”)

[0347] water, to 5 ml final volume

[0348] centrifuge sample 5 min at room temperature, 20,000×g to pelletinsoluble aggregates

[0349] load sample onto tube gel

[0350] apply overlay solution

[0351] 1.9 g urea (3 M final)

[0352] 0.67 ml 10% Triton X-100 (0.67% final)

[0353] 167 μl β-mercaptoethanol (1.67% final)

[0354] 133 μl ampholytes pH 5-7 (0.53% final)

[0355] 33 μl ampholytes pH 3-10 (0.12% final)

[0356] add a few grains of bromophenol blue

[0357] water, to 10 ml final volume

[0358] gently overlay the overlay with de-gassed 20 mM NaOH, beingcareful not to disturb sample

[0359] fill lower buffer chamber (anode) with 20 mM H₃PO₄

[0360] place gel in tank and fill upper chamber (cathode) with de-gassed20 mM NaOH

[0361] connect power supply and apply voltage, using a constant powersetting of 3 watts, limit voltage to 600 V

[0362] Measure pH of IEF Gel for Extra 1-D Gel

[0363] set up 24 tubes, each containing 1 ml high purity, de-gassedwater

[0364] briefly rinse IEF gel in water

[0365] place gel on glass plate and dissect into 24×0.5 cm pieces

[0366] place gel pieces, in order, into tubes

[0367] soak in water on shaker for 2-3 hr

[0368] read pH of each tube

[0369] Stain IEF Gels with Coomassie or Silver

[0370] stain one gel with Coomassie Blue overnight; destain

[0371] fix other gel in 30% ethanol/10% acetic acid for silver staining

[0372] silver stain: follow protocol in Molecular Cloning Lab Manual(Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Laboratory, N.Y.).

[0373] Run Second Dimension Laemmli gels

[0374] pour 7.5% Laemmli gels, using 3 mm spacers and comb to generateone long well (˜13 cm)

[0375] equilibrate each tube gel in 15 ml equilibration buffer for 5-7min

[0376] 3% (w/v) SDS

[0377] 0.4 mM EDTA

[0378] 10% (v/v) glycerol

[0379] 20 mM TrisHCl, pH 6.8

[0380] 1.7% 2-mercaptoethanol

[0381] bromophenol blue

[0382] fill Laemmli gel well with SDS-PAGE running buffer

[0383] place IEF gel in well of Laemmli gel (acidic end to theleft/basic end to the right)

[0384] drain buffer from well and carefully overlay IEF strip withagarose/equilibration buffer mixture

[0385] make 2% (w/v) L.M.P. agarose, melt and keep at 40° C.

[0386] mix 1:1 with equilibration buffer

[0387] also mix 50 μl MW standards with 50 μl agarose/equilibrationbuffer and load into MW lanes

[0388] after agarose has set, assemble electrophoresis tank, overlaygels with SDS-PAGE tank buffer and run at constant current (15 mA/gel)for 5 hr

[0389] turn down to 8 mA/gel overnight (16 hr)

[0390] turn up to 30 mA/gel for final 6 hr

[0391] Electrophoretic Protein Transfer (One 2-D Gel)

[0392] pre-wet immobilon PVDF membrane in 100% methanol

[0393] transfer proteins 20 hr @ 100 mA (˜2 Amp-hr) in 1×Towbin+5%Methanol at 4° C.

[0394] block blots in HBS+5% milk+1% goat serum+0.1% NaN₃ at room tempfor 1 hr

[0395] Stain Blot with Phage-cDNA Library/Ab Reagent as Above for 1-DGels

[0396] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent or patent application werespecifically and individually indicated to be so incorporated byreference.

What is claimed is:
 1. A method of analyzing a polypeptide, comprising:(a) providing a population of replicable genetic package/antibodyreagents, wherein members of the population comprise a replicablegenetic package that displays a first polypeptide encoded by aheterologous segment of a nucleic acid of the package, and the firstpolypeptide is complexed with a multivalent captured antibody havingspecific affinity for the first polypeptide; the first polypeptide andthe captured antibody complexed with it varying between at least some ofthe package/antibody reagents; (b) contacting the population ofpackage/antibody reagents with a second polypeptide, wherebypackage/antibody reagents bearing captured antibodies having specificaffinity for the second polypeptide bind to the second polypeptide; and(c) identifying at least one package/antibody reagent that binds to thesecond polypeptide; and (d) determining the sequence of the segment ofthe nucleic acid of the at least one package/antibody reagent and thecorresponding amino acid sequence as an indication of an epitope sharedby the first and second polypeptide.
 2. The method of claim 1, whereinthe amino acid sequence is an indication of an amino acid sequence of anepitope on the second polypeptide.
 3. The method of claim 1, wherein theproviding step (a) comprises (i) cloning cDNA molecules prepared from acell or tissue into a vector to create the replicable genetic packages;and (ii) incubating a population of antibodies with the replicablegenetic packages to form the population of package/antibody reagents. 4.The method of claim 3, wherein the preparing step (a) further comprisespreparing a population of immunogens and generating the population ofantibodies with the population of immunogens.
 5. The method of claim 4,wherein the population of immunogens comprise at least some of theproteins present in the cell or tissue.
 6. The method of claim 5,wherein the at least some of the proteins are part of a fusion protein.7. The method of claim 6, wherein the fusion protein includesglutathione-S-transferase (GST).
 8. The method of claim 4, wherein thepopulation of immunogens comprises a display library, the members of thedisplay library comprising a replicable genetic package that displaysone of the first polypeptides displayed by the package/antibodyreagents.
 9. The method of claim 8, wherein the polypeptides displayedon members of the display library are peptides having random amino acidsequences.
 10. The method of claim 9, wherein the replicable geneticpackages which display the peptides differ in type from the replicablegenetic packages of the package/antibody reagents.
 11. The method ofclaim 9, wherein the peptides displayed on members of the displaylibrary are up to 100 amino acids in length.
 12. The method of claim 11,wherein the peptides displayed on members of the display library are 6to 20 amino acids in length.
 13. The method of claim 12, wherein thepeptides displayed on members of the display library are 6 to 10 aminoacids in length.
 14. The method of claim 1, wherein (i) the secondpolypeptide is one of multiple different polypeptides within a sample;(ii) the contacting step (b) comprises contacting the population ofpackage/antibody reagents with the sample; and (iii) the identifyingstep (c) comprises identifying package/antibody reagents that haveformed complexes with the different polypeptides.
 15. The method ofclaim 1, wherein the second polypeptide is within an electrophoreticseparation matrix, and the contacting step (b) comprises excising asection from the separation matrix that contains the second polypeptide,eluting the second polypeptide from the excised section and contactingthe eluted second polypeptide with the package/antibody reagents. 16.The method of claim 1, wherein (i) the second polypeptide is apolypeptide located within an electrophoretic separation matrix; (ii)the contacting step (b) comprises contacting the separation matrix or areplica thereof with the package/antibody reagents; and (iii) theidentifying step (c) comprises isolating the at least onepackage/antibody reagent from the separation matrix or the replicathereof.
 17. The method of claim 1, wherein (i) the second polypeptideis one of a plurality of polypeptides at different locations within atwo-dimensional electrophoretic separation matrix; (ii) the contactingstep (b) comprises contacting the two-dimensional electrophoreticseparation matrix or a replica thereof with the package/antibodyreagents; (iii) the identifying step (c) comprises isolatingpackage/antibody reagents that have formed complexes with the differentpolypeptides; and (iv) the determining step (d) comprises determiningthe sequence of the segment of the nucleic acid of each of the isolatedpackage/antibody reagents.
 18. The method of claim 17, wherein (i) theplurality of polypeptides within the separation matrix comprise aplurality of polypeptides that have different primary sequences and aplurality of polypeptides that have the same primary sequence but whichare differentially modified; and (ii) the determining step (d) comprisesdetermining which of the plurality of proteins within the separationmatrix have the same primary sequence but are differentially modified.19. The method of claim 1, wherein (i) the providing step (a) comprisesproviding an array in which the population of package/antibody reagentsare immobilized on a support; and (ii) the contacting step (b) comprisesapplying a sample containing the second polypeptide to the array. 20.The method of claim 19, wherein (i) the second polypeptide is one ofmultiple different polypeptides within the sample; (ii) the identifyingstep (c) comprises identifying package/antibody reagents that haveformed complexes to the different polypeptides; and (iii) thedetermining step (d) comprises determining the sequence of the segmentof the nucleic acid for each of the package/antibody reagents that arecomplexed to the different polypeptides.
 21. The method of claim 19,further comprising preparing the array, the preparing step comprisingspotting the package/antibody reagents onto the support.
 22. The methodof claim 19, further comprising preparing the array, the preparing stepcomprising (i) growing bacterial colonies that express the replicablegenetic packages of the package/antibody reagents; (ii) transferringcolonies onto the support; and (iii) contacting the support with thecaptured antibodies to form the array.
 23. The method of claim 19,wherein the replicable genetic packages of the package/antibody reagentsare lytic bacteriophage and the method further comprises preparing thearray, the preparing step comprising (i) growing the bacteriophage on alawn of bacteria to produce a plurality of plaques; (ii) transferringplaques to the support; and (iii) contacting the support with thecaptured antibodies to form the array.
 24. The method of claim 19,wherein the second polypeptide is labeled and the identifying step (c)comprises detecting labeled second polypeptide complexed to the at leastone package/antibody reagent.
 25. The method of claim 19, wherein (i)the contacting step (b) further comprises contacting the array with oneor more labeled detection reagents, whereby a labeled detection reagentwith specific affinity for the second polypeptide binds to the secondpolypeptide of the at least one package/antibody reagent to form atertiary complex comprising the at least one package/antibody complex,the second polypeptide and the detection reagent; and (ii) theidentifying step (c) comprises detecting the tertiary complex, therebyidentifying the at least one package/antibody complex.
 26. The method ofclaim 25, wherein the one or more detection reagents comprise adetection antibody with specific affinity for the second polypeptide.27. The method of claim 25, wherein (i) the one or more detectionreagents comprise a replicable genetic package which has a copy of thesame nucleic acid as the package/antibody reagent that complexes withthe second polypeptide, and which is complexed to a detection antibodyhaving specific affinity for the second polypeptide; and (ii) thedetermining step (d) comprises determining the sequence of the segmentof the nucleic acid of the replicable genetic package of the detectionreagent, thereby providing an indication of the sequence of the segmentof the nucleic acid of the at least one package/antibody reagent. 28.The method of claim 27, wherein the label is attached to the replicablegenetic package of the detection reagent.
 29. The method of claim 27,wherein the determining step (d) further by comprises retrieving thereplicable genetic package of the detection reagent from the tertiarycomplex, amplifying the segment of the nucleic acid of the detectionreagent and sequencing the amplified segment, the sequence of theamplified segment corresponding to the sequence of the segment of the atleast one package/antibody reagent.
 30. The method of claim 29, whereinretrieval of the package/antibody reagent from the detection reagent isperformed with a microdissection instrument.
 31. The method of claim 30,wherein the microdissection instrument is a laser capturemicrodissection instrument.
 32. The method of claim 19, wherein thedetermining step (d) further comprises retrieving the at least onepackage/antibody reagent from the array, amplifying the segment of thenucleic acid of the at least one package/antibody reagent and sequencingthe amplified segment.
 33. The method of claim 24, the method furthercomprising preparing an archival replica of the array, and wherein thedetermining step (d) further comprises isolating a replicable geneticpackage from the archival replica that corresponds to the at least onepackage/antibody reagent, and determining the sequence of the segment ofthe nucleic acid in the isolated replicable genetic package, therebydetermining the sequence of the segment of the nucleic acid in the atleast one package/antibody reagent.
 34. The method of claim 25, themethod further comprising preparing an archival replica of the array,and wherein the determining step (d) further comprises isolating areplicable genetic package from the archival replica that corresponds tothe at least one package/antibody reagent, and determining the sequenceof the segment of the nucleic acid in the isolated replicable geneticpackage, thereby determining the sequence of the segment of the nucleicacid in the at least one package/antibody reagent.
 35. The method ofclaim 1, wherein the second polypeptide is obtained from or contained ina tissue sample.
 36. The method of claim 35, wherein the tissue sampleis a slice from a tissue.
 37. The method of claim 1, wherein the secondpolypeptide is obtained from or contained in a subcellular compartment.38. The method of claim 1, wherein the replicable genetic package of thepackage/antibody complex is selected from the group consisting of avirus, a bacteriophage, a bacterium, a polysome and a spore.
 39. Themethod of claim 1, wherein the replicable genetic package is abacteriophage.
 40. The method of claim 1, wherein the capturedantibodies comprise monovalent antibodies displayed in a multivalentformat.
 41. The method of claim 40, wherein the monovalent antibodiesare scFv polypeptides.
 42. The method of claim 41, wherein the scFvpolypeptides are displayed in a multivalent format on phage.
 43. Themethod of claim 1, wherein the captured antibody is a diabody, a tribodyor a tetrabody.
 44. The method of claim 1, wherein at least some of themembers of the population display a single copy of the first polypeptidewhich is complexed with a single copy of the captured antibody.
 45. Themethod of claim 1, wherein the first polypeptide is complexed with aplurality of copies of the same captured antibody.
 46. The method ofclaim 1, wherein the first polypeptide is complexed with multipledifferent antibodies.
 47. The method of claim 1, wherein at least someof the members of the population display multiple copies of the firstpolypeptide, each copy of the first polypeptide complexed with one ormore captured antibodies having specific affinity for the firstpolypeptide, whereby such members display a plurality of antibodies. 48.The method of claim 47, wherein at least some of the plurality ofantibodies have different protein sequences but specific affinity forthe same epitope.
 49. The method of claim 47, wherein at least some ofthe plurality of antibodies have different protein sequences and havespecific affinity for different epitopes.
 50. The method of claim 1,wherein the contacting step comprises contacting at least some membersof the population to form an aggregate, an aggregate comprising aplurality of members linked via an antibody that is complexed to a firstpolypeptide on each of the plurality of members.
 51. The method of claim1, wherein (i) the second polypeptide with which the population ofpackage/antibody complexes is contacted is a receptor expressed on asurface of a cell, whereby the at least one package/antibody complexbinds to the receptor and is subsequently transported into and/orthrough the cell; and (ii) the identifying step comprises detecting thetransport of the at least one package/antibody reagent into and/orthrough the cell.
 52. The method of claim 51, wherein the cell is apolarized cell and the identifying step comprises detecting transportthrough the cell.
 53. The method of claim 51, wherein the cell is anon-polarized cell and the identifying step comprises detectingtransport into the cell.
 54. A method of analyzing a sample containing amixture of polypeptides, the method comprising: (a) providing an arraycomprising a support and a plurality of replicable geneticpackage/antibody reagents immobilized to the support, wherein thepackage/antibody reagents comprise a replicable genetic package thatdisplays a polypeptide encoded by a segment of a nucleic acid of thepackage, and the polypeptide is complexed with a captured antibodyhaving specific affinity for the polypeptide, the polypeptide and thecaptured antibody complexed with it varying between at least some of thepackage/antibody reagents; (b) contacting the array with the samplecontaining the mixture of polypeptides, whereby package/antibodyreagents bearing captured antibodies having specific affinity for apolypeptide in the mixture capture the polypeptide from the mixture toform a complex; and (c) detecting at least one of the complexes, thesequence of the segment of the nucleic acid of the package/antibodyreagent within the at least one complex and the corresponding amino acidsequence providing an indication of an amino acid sequence of an epitopeon the captured polypeptide.
 55. The method of claim 54, furthercomprising repeating steps (a)-(c) with a second array and a secondsample and comparing which of the package/antibody reagents of the twoarrays form complexes with the polypeptides in the samples.
 56. Themethod of claim 54, further comprising quantifying the amount ofpolypeptide in the complex(es) detected in step (c).
 57. The method ofclaim 54, further comprising differentially labeling polypeptides in twosamples of proteins, such that polypeptides in one sample bear one labeland polypeptides in the other sample bear a different label, and wherein(i) the contacting step (b) comprises contacting the array with the twosamples, whereby package/antibody reagents bearing captured antibodieshaving specific affinity for a polypeptide in the samples capture thepolypeptide to form a complex; and (ii) the detecting step (c) comprisesdetecting differentially labeled complexes on the array.
 58. Areplicable genetic package reagent, comprising a replicable geneticpackage that displays a polypeptide encoded by a heterologous segment ofa nucleic acid of the package, and the polypeptide is complexed with amultivalent captured antibody having specific affinity for thepolypeptide.
 59. A collection of replicable genetic package/antibodyreagents, wherein each member of the collection comprises a replicablegenetic package that displays a polypeptide encoded by a heterologoussegment of a nucleic acid of the package, and the polypeptide iscomplexed with a multivalent captured antibody having specific affinityfor the polypeptide, the polypeptide and the captured antibody complexedwith it varying between the package/antibody reagents.
 60. Thecollection of claim 59, wherein the replicable genetic packages areselected from the group consisting of a virus, a bacteriophage, abacterium, a polysome and a spore.
 61. The collection of claim 60,wherein the replicable genetic packages are bacteriophage.
 62. Thecollection of claim 58, wherein the captured antibody comprises aplurality of monovalent antibodies displayed in a multivalent format.63. The collection of claim 59, wherein members of the collection arelabeled.
 64. The collection of claim 63, wherein the replicable geneticpackages are bacteriophage and the label is attached to thebacteriophage.
 65. The collection of claim 59, wherein the monovalentantibodies are scFv polypeptides.
 66. The collection of claim 59,wherein the captured antibody is a diabody, a tribody or a tetrabody.67. The collection of claim 59, wherein at least some of the members ofthe collection display a single copy of the polypeptide, and the singlepolypeptide is complexed with a single copy of the captured antibody.68. The collection of claim 59, wherein the polypeptide is complexedwith a plurality of captured antibodies.
 69. The collection of claim 59,wherein at least some of the members of the population display multiplecopies of the polypeptide, each copy of the polypeptide complexed withone or more captured antibodies having specific affinity for thepolypeptide, whereby such members display a plurality of antibodies. 70.The collection of claim 69, wherein at least some of the plurality ofantibodies have different protein sequences but specific affinity forthe same epitope.
 71. The collection of claim 69, wherein at least someof the plurality of antibodies have different protein sequences and havespecific affinity for different epitopes.
 72. The collection of claim59, wherein the package/antibody reagents are immobilized to a supportto form an array.
 73. The collection of claim 72, wherein there are atleast 100 different package/antibody reagents, each displaying acaptured antibody having specific binding affinity for a differentprotein.
 74. The collection of claim 73, wherein there are at least 1000different package/antibody reagents, each displaying a captured antibodyhaving specific binding affinity for a different protein.
 75. Thecollection of claim 72, wherein the package/antibody reagents displaycaptured antibodies having specific binding affinity for at least 80% ofthe expressed proteins in a cell or tissue.
 76. The collection of claim72, wherein at least some of the multivalent captured antibodies of thepackage/antibody reagents are further complexed with a capturedpolypeptide.
 77. The collection of claim 76, wherein the capturedpolypeptide is a functional polypeptide.
 78. An array of polypeptides,comprising (a) a support; and (b) a plurality of polypeptidesimmobilized at different locations on the support, wherein there are atleast 10³ locations/cm² on the support, each location having at leastone of the plurality of polypeptides immobilized therein, and whereinpolypeptides in at least some of the locations differ in amino acidsequence and/or another property from polypeptides in other locations.79. The array of claim 78, wherein there are at least 10⁴ locations/cm².80. The array of claim 79, wherein there are at least 10⁶ locations/cm².81. The array of claim 80, wherein there are at least 10⁸ locations/cm².82. The array of claim 81, wherein there are at least 10¹⁰locations/cm².
 83. The array of claim 79, wherein there are at least 100different polypeptides immobilized to the support.
 84. The array ofclaim 83, wherein there are at least 1000 different polypeptidesimmobilized to the support.
 85. The array of claim 84, wherein there areat least 10,000 different polypeptides immobilized to the support. 86.The array of claim 85, wherein there are at least 100,000 differentpolypeptides immobilized to the support.
 87. The array of claim 78,wherein the at least one polypeptide immobilized at each locationdiffers in sequence and/or other property from the at least onepolypeptide at each of the other locations.
 88. The array of claim 78,wherein the polypeptides are functional polypeptides.
 89. The array ofclaim 88, wherein each of the functional polypeptides is complexed to acaptured antibody that is bound to a polypeptide displayed on areplicable genetic package that is immobilized to the support.
 90. Thearray of claim 78, wherein the polypeptides are antibodies.
 91. Thearray of claim 90, wherein each of the antibodies is complexed to apolypeptide displayed on a replicable genetic package that isimmobilized to the support.
 92. The array of claim 91, wherein theantibodies are bound to polypeptides displayed on phage which areimmobilized to the support, and the array is prepared by (i) platingphage on a layer of cells to form bacterial microcolonies or an array ofmicro-plaques; (ii) replicating the microcolonies or plaques onto thesupport, whereby phage displaying polypeptides become immobilized to thesupport; and (iii) contacting the displayed polypeptides with apopulation of antibodies under conditions such that antibodies formcomplexes with displayed polypeptides for which the antibodies havespecific binding affinity, whereby the array of antibodies is formed.93. An array, comprising (a) a support; and (b) a plurality ofpolypeptides immobilized to the support, at least some of the pluralityof polypeptides complexed with a captured antibody of a package/antibodyreagent, each package/antibody reagent comprising a replicable geneticpackage that displays a polypeptide that is complexed to the capturedantibody.
 94. The array of claim 93, wherein the support is a gel or areplica thereof, and the plurality of polypeptides are located withinthe gel or on the replica.
 95. A method for preparing an array ofpolypeptides, the method comprising: (a) immobilizing replicable geneticpackages displaying a polypeptide to a support; and (b) contacting thedisplayed polypeptides with a population of antibodies under conditionssuch that the antibodies form complexes with displayed polypeptides forwhich the antibodies have specific binding affinity, whereby the arrayof polypeptides is formed.
 96. The method of claim 95, wherein thereplicable genetic packages are phage and immobilizing comprises platingthe phage on a layer of cells to form bacterial microcolonies or anarray of micro-plaques; and replicating the microcolonies ormicro-plaques onto the support, whereby phage displaying polypeptidesbecome immobilized to the support.
 97. The method of claim 95, furthercomprising contacting the array with a sample containing a plurality ofproteins under conditions such that proteins in the sample andantibodies on the array that have specific binding affinity for oneanother form complexes, thereby forming an array of captured proteins.98. The method of claim 97, wherein the plurality of proteins in thesample are functional proteins.